Protein production in filamentous fungal cells in the absence of inducing substrates

ABSTRACT

Certain embodiments of the disclosure are directed to variant filamentous fungal cells, compositions thereof and methods thereof for increased production of one or more proteins of interest. More particularly, in certain embodiments, the disclosure is directed to variant filamentous fungal (host) cells derived from parental filamentous fungal cells, wherein the variant host cells comprise a genetic modification which enables the expression/production of a protein of interest (POI) in the absence of inducing substrate. In certain embodiments, a variant fungal host cell of the disclosure comprises a genetic modification which increases the expression of a variant activator of cellulase expression 3 (ace3) gene encoding an Ace3 protein referred to herein as Ace3-L.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.16/336,500, filed Mar. 26, 2019, which is a 371 of InternationalApplication No. PCT/US2017/54983, filed Oct. 3, 2017, which claimsbenefit to U.S. Provisional Application No. 62/403,787, filed Oct. 4,2016, all of which are hereby incorporated by reference in theirentirety.

SEQUENCE LISTING

The sequence listing text file submitted herewith via EFS contains thefile “NB41159WOPCT_SEQLISTING.txt” created on Oct. 2, 2017, which is 157kilobytes in size. This sequence listing complies with 37 C.F.R. §1.52(e) and is incorporated herein by reference in its entirety.

FIELD

The present disclosure is generally related to the fields of molecularbiology, biochemistry, protein production and filamentous fungi. Certainembodiments of the disclosure are directed to variant filamentous fungalcells, compositions thereof and methods thereof for increased productionof one or more proteins of interest. More particularly, in certainembodiments, the disclosure is directed to variant filamentous fungal(host) cells derived from parental filamentous fungal cells, wherein thevariant host cells comprise a genetic modification which enables theexpression/production of one or more proteins of interest (POI) in theabsence of inducing substrate.

BACKGROUND

Cellulose, a component of lignocellulosic plant material, is the mostabundant polysaccharide found in nature. Likewise, filamentous fungi areknown in the art to be efficient degraders of plant biomass, and are infact a major source of industrially relevant lignocellulosic degradingenzymes (referred to hereinafter collectively as “cellulase” enzymes).For example, filamentous fungi are known to produce extracellularcellulase enzymes (e.g., cellobiohydrolases, endoglucanases,β-glucosidases) that hydrolyze the β-(1,4)-linked glycosidic bonds ofcellulose to produce glucose (i.e., thereby conferring the ability ofthese filamentous fungi to utilize cellulose for growth).

In particular, the filamentous fungus Trichoderma reesei (T. reesei; ananamorph of the fungus Hypocrea jecorina) is known to be an efficientproducer of cellulase enzymes (see, e.g., PCT International ApplicationNos. WO1998/15619, WO2005/028636, WO2006/074005, WO1992/06221,WO1992/06209, WO1992/06183, WO2002/12465 and the like). As such,filamentous fungi such as T. reesei have been utilized for their abilityto produce enzymes which are valuable in the production of suchcommodities as cellulosic derived ethanol, textiles and clothing,detergents, fibers, food and feed additives and other industrial uses.

The expression (and production) of these industrially relevant enzymesin Trichoderma are known to be dependent on the carbon source availablefor growth. More particularly, the production of cellulase enzymes byfilamentous fungi is an energy-consuming process and as such, bothinducing and repressing mechanisms have evolved in filamentous fungi toensure the efficient production of these enzymes. For example, thevarious genes encoding enzymes needed for the degradation of plant cellwall material (i.e., cellulases/hemicellulases) are “activated” in thepresence of an “inducing” substrate and “repressed” in the presence ofeasily metabolized carbon sources (e.g., D-glucose) that are preferredover plant biomass via a mechanism known as “carbon cataboliterepression” (hereinafter, “CCR”). Thus, the cellulase genes are tightlyrepressed by glucose and are induced several thousand folds by celluloseand certain disaccharides (e.g., sophorose, lactose, gentiobiose). Forexample, the expression level of the major cellobiohydrolase 1 (cbh1) is“up-regulated” several thousand fold on media containing inducing carbonsources such as cellulose or sophorose, compared with glucose containingmedia (Ilmen et al., 1997). Furthermore, the addition of a “repressing”carbon source to “induced” T. reesei cultures was shown to override the(cellulose or sophorose) induction, thereby resulting in down-regulationof cellulase gene expression (el-Gogary et al., 1989; Ilmen et al.,1997). Thus, the expression of genes comprising the cellulase system(enzymes) are at least coordinated and regulated at the transcriptionallevel, wherein the gene members of this system act synergistically, andas noted above, are necessary for the efficient hydrolysis of celluloseto soluble oligosaccharides.

More specifically, a genome-wide analysis revealed that there are atleast ten (10) cellulolytic and sixteen (16) xylanolytic enzyme encodinggenes in T. reesei (Martinez et al., 2008). In particular, the mostabundantly secreted enzymes are the two main cellobiohydrolases (EC.3.2.1.91), cbh1 (cellobiohydrolase 1) and cbh2 (cellobiohydrolase 2),and the two major specific endo-β-1,4-xylanases (EC 3.3.1.8), xyn1(endo-1,4-beta-xylanase 1) and xyn2 (endo-1,4-beta-xylanase 2), referredto herein as “major industrially relevant hemicellulases and cellulases”or “MIHCs”. These MIHCs work together with additional enzymes to degradecellulose and xylan, which results in the formation of solubleoligosaccharides and monosaccharides, such as cellobiose, D-glucose,xylobiose and D-xylose. In addition, sophorose is a product of thetransglycosylation activity of some of these enzymes (Vaheri et al.,1979). More particularly, these soluble oligo- and monosaccharides(i.e., cellobiose, D-glucose, xylobiose, D-xylose and sophorose) havebeen reported in the literature to influence the expression of the MICHsin T. reesei. For example, the presence of D-glucose causes CCR, whichresults in the secretion of low quantities of MIHCs. Sophorose isbelieved to be the most potent inducer for the expression of cbh1 andcbh2. D-xylose modulates xyn1 and xyn2 expression in a concentrationdependent manner.

In general, the commercial scale production of enzymes/polypeptides byfilamentous fungi such as Trichoderma is typically by either solid orsubmerged culture, including batch, fed batch, and continuous flowprocesses. For example, one of the most problematic and expensiveaspects of industrial cellulase production in Trichoderma is providingthe appropriate inducer (i.e., inducing substrate) to the Trichodermahost cells. For example, as is the case for laboratory scaleexperiments, cellulase (enzyme) production on a commercial scale is“induced” by growing the fungal cells on solid cellulose (i.e., aninducing substrate) or by culturing the cells in the presence of adisaccharide inducer such as “lactose” (i.e., an inducing substrate).

Unfortunately, on an industrial scale, both methods of “induction” havedrawbacks which result in high costs being associated with cellulaseproduction. For example, as set forth above, cellulase synthesis issubject to both cellulose induction and glucose repression. Thus, acritical factor influencing the yield of cellulase enzymes under thecontrol of an inducible promoter is the maintenance of a proper balancebetween cellulose substrate and glucose concentration (i.e., it iscritical for obtaining reasonable commercial yields of the regulatedgene product). Although cellulose is an effective and inexpensiveinducer, controlling the glucose concentration when filamentous fungalcells are grown on solid cellulose can be problematic. At lowconcentrations of cellulose, glucose production may be too slow to meetthe metabolic needs of active cell growth and function. On the otherhand, cellulase synthesis can be halted by glucose repression whenglucose generation is faster than its consumption. Thus, expensiveprocess control schemes are required to provide slow substrate additionand monitoring of glucose concentration (Ju and Afolabi, 1999).Moreover, the slow continuous delivery of substrate can be difficult toachieve as a result of the solid nature of the cellulosic materials.

Some of the problems associated with the use of cellulose as an“inducing substrate” can be overcome through the use of soluble“inducing substrates” such as “lactose”, “sophorose” or “gentiobiose”.For example, when using lactose as an “inducing substrate”, the lactosehas to be provided at high concentrations so as to function as aninducer and a carbon source (e.g., see Seiboth, et. al., 2002).Sophorose is a more potent inducer than cellulose, but sophorose isexpensive and difficult to manufacture. For example, a mixture ofglucose, sophorose and other disaccharides (i.e., generated viaenzymatic conversion of glucose) can be used for the efficientproduction of cellulases, which incurs a greater (production) cost thanusing glucose alone. Thus, while it is easier to handle and control thansolid cellulose, the use of sophorose as an inducing substrate cannonetheless make the cost of producing cellulases prohibitivelyexpensive.

Based on the foregoing, it is evident that there remains an ongoing andunmet need in the art for cost effective commercial scale production ofenzymes/polypeptides by filamentous fungi without the need orrequirement of providing costly inducing substrates (e.g., sophorose,lactose and the like) for such production. More particularly, thereremains a need in the art for the commercial scale production of one ormore endogenous lignocellulosic degrading enzymes by filamentous fungalhost cells, wherein such filamentous fungal cells are capable ofexpressing one or more of these genes in the absence of an inducingsubstrate. In addition, there are further unmet needs in the art forcost effective production of one or more heterologous protein productsexpressed and produced in such filamentous fungal host cells, whereinthe heterologous genes encoding such proteins are introduced into afungal host cell, which is capable of expressing such heterologous genesin the absence of an inducing substrate.

BRIEF SUMMARY

Certain embodiments of the disclosure are related to the commercialscale production of enzymes/polypeptides by filamentous fungi withoutthe need or requirement of providing costly inducing substrates (e.g.,sophorose, lactose and the like) for such production. Thus, certainother embodiments are related to variant filamentous fungal cells,compositions thereof and methods thereof for increased production of oneor more proteins of interest. For example, certain embodiments of thedisclosure are directed to a variant filamentous fungal cell derivedfrom a parental filamentous fungal cell, the variant cell comprising anintroduced polynucleotide construct encoding an Ace3 protein comprisingabout 90% sequence identity to an Ace3 protein of SEQ ID NO: 6, whereinthe variant cell produces an increased amount of a protein of interest(POI) in the absence of an inducing substrate relative to the parentalcell, wherein the variant and parental cells are cultivated undersimilar conditions. In certain other embodiments, the variant cellproduces an increased amount of a POI in the presence of an inducingsubstrate relative to the parental cell, wherein the variant andparental cells are cultivated under similar conditions.

In another embodiment of the variant cell, an encoded Ace3 proteincomprising about 90% sequence identity to SEQ ID NO: 6, comprises“Lys-Ala-Ser-Asp” as the last four C-terminal amino acids. In anotherembodiment, an encoded Ace3 protein comprising about 90% sequenceidentity to SEQ ID NO: 6, comprises an N-terminal amino acid fragment ofSEQ ID NO: 98 operably linked and preceding SEQ ID NO: 6. In certainother embodiments of the variant cell, the Ace-3 protein comprises about90% sequence identity to SEQ ID NO: 12. In another embodiment, theintroduced polynucleotide encoding an Ace3 protein comprises an openreading frame (ORF) sequence comprising about 90% identity to SEQ ID NO:5.

In yet other embodiments of the variant cell, the POI is an endogenousPOI or a heterologous POI. Thus, in certain embodiments, the variantcell comprises an introduced polynucleotide construct encoding aheterologous POI. In another embodiment, a polynucleotide constructencoding the heterologous POI is expressed under the control of acellulose-inducible gene promoter. In certain other embodiments, anendogenous POI is a lignocellulose degrading enzyme. Thus, in otherembodiments, a lignocellulose degrading enzyme is selected from thegroup consisting of cellulase enzymes, hemi-cellulase enzymes, or acombination thereof. In certain other embodiments, a lignocellulosedegrading enzyme is selected from the group consisting of cbh1, cbh2,egl1, egl2, egl3, egl4, egl5, egl6, bgl1, bgl2, xyn1, xyn2, xyn3, bxl1,abf1, abf2, abf3, axe1, axe2, axe3, man1, agl1, agl2, agl3, gir1, swo1,cip1 and cip2. In yet other embodiments, a heterologous POI is selectedfrom the group consisting of an α-amylase, an alkaline α-amylase, aβ-amylase, a cellulase, a dextranase, an α-glucosidase, anα-galactosidase, a glucoamylase, a hemicellulase, a pentosanase, axylanase, an invertase, a lactase, a naringanase, a pectinase, apullulanase, an acid protease, an alkali protease, a bromelain, aneutral protease, a papain, a pepsin, a peptidase, a rennet, a rennin, achymosin, a subtilisin, a thermolysin, an aspartic proteinase, atrypsin, a lipase, an esterase, a phospholipase, a phosphatase, aphytase, an amidase, an iminoacylase, a glutaminase, a lysozyme, apenicillin acylase; an isomerase, an oxidoreductases, a catalase, achloroperoxidase, a glucose oxidase, a hydroxysteroid dehydrogenase, aperoxidase, a lyase, an aspartic β-decarboxylase, a fumarase, ahistadase, a transferase, a ligase, an aminopeptidase, acarboxypeptidase, a chitinase, a cutinase, a deoxyribonuclease, anα-galactosidase, a β-galactosidase, a β-glucosidase, a laccase, amannosidase, a mutanase, a polyphenol oxidase, a ribonuclease and atransglutaminase.

In another embodiment of the variant cell, the polynucleotide constructcomprises a promoter sequence 5′ and operably linked to thepolynucleotide sequence encoding an Ace3 protein comprising about 90%sequence identity to SEQ ID NO: 6. In certain embodiments, thepolynucleotide construct further comprises a native ace3 terminatorsequence 3′ and operably linked to the polynucleotide sequence encodingan Ace3 protein comprising about 90% sequence identity to SEQ ID NO: 6.In certain other embodiments, the polynucleotide construct is integratedinto the fungal cell genome. In certain embodiments, the polynucleotideconstruct is integrated into a telomere site of the fungal cell genome.In certain other embodiments, the polynucleotide construct is integratedinto a glucoamylase (gla1) gene locus of the fungal cell genome. In yetother embodiments, the polynucleotide construct comprises a nucleotidesequence comprising about 90% sequence identity to SEQ ID NO: 4, SEQ IDNO: 11 or SEQ ID NO: 13. In other embodiments, an encoded Ace3 proteincomprises an amino acid sequence comprising 95% sequence identity to SEQID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 14. Thus, in other embodiments, anencoded Ace3 protein comprises an amino acid sequence of SEQ ID NO: 6,SEQ ID NO: 12 or SEQ ID NO: 14.

In other embodiments, the variant cell comprises a genetic modificationwhich expresses a polynucleotide encoding a wild-type xylanase regulator1 (Xyr1) protein of SEQ ID NO: 48 or a variant xylanase regulator 1(Xyr1) protein of SEQ ID NO: 46. In certain other embodiments, thevariant cell comprises a genetic modification which reduces or preventsthe expression of a gene encoding an endogenous carbon cataboliterepressor 1 (Cre1) protein or an Ace1 protein. In yet anotherembodiment, the variant cell comprises a genetic modification whichcomprises expressing an Ace2 protein. In other embodiments, thefilamentous fungal cell is a Pezizomycotina cell of the Ascomycotasubphylum. In certain other embodiments, the filamentous fungal cell isa Trichoderma sp. cell.

In other embodiments, the disclosure is related a polynucleotide ORFencoding an Ace3 protein comprising about 90% sequence identity to SEQID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 14.

In other embodiments, the disclosure is related a lignocellulosedegrading enzyme produced by the variant cell of the disclosure. Inother embodiments, the disclosure is related a heterologous POI producedby the variant cell of cell of the disclosure.

In other embodiments, the disclosure is related to a method forproducing an endogenous protein of interest in a Trichoderma sp. fungalcell in the absence of an inducing substrate, the method comprising (i)introducing into the fungal cell a polynucleotide construct comprisingin the 5′ to 3′ direction (a) a first nucleic acid sequence comprising apromoter and (b) a second nucleic acid sequence operably linked to thefirst nucleic acid sequence, wherein the second nucleic acid sequenceencodes an Ace3 protein comprising about 90% sequence identity to SEQ IDNO: 6 and comprises “Lys-Ala-Ser-Asp” as the last four C-terminal aminoacids, and (ii) fermenting the cells of step (i) under conditionssuitable for fungal cell growth and protein production, wherein suchsuitable growth conditions do not include an inducing substrate. Incertain embodiments of the method, the polynucleotide constructcomprises a third nucleic acid sequence 3′ and operably linked to thesecond nucleic acid, wherein the third nucleic acid sequence comprises anative ace3 terminator sequence. In another embodiment, thepolynucleotide construct is integrated into the fungal cell genome. Incertain embodiments, the polynucleotide construct is integrated into atelomere site of the fungal cell genome. In certain other embodiments,the polynucleotide construct is integrated into a glucoamylase (gla1)gene locus of the fungal cell genome. In other embodiments of themethod, the polynucleotide construct comprises a nucleotide sequencecomprising about 90% sequence identity to SEQ ID NO: 4, SEQ ID NO: 11 orSEQ ID NO: 13. In other embodiments, the encoded Ace3 protein comprisesan amino acid sequence comprising 95% sequence identity to SEQ ID NO: 6,SEQ ID NO: 12 or SEQ ID NO: 14. In another embodiment, the encoded Ace3protein comprises an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 12or SEQ ID NO: 14.

In certain embodiments of the method, the step (i) promoter is selectedfrom the group consisting of a rev3 promoter (SEQ ID NO: 15), a bxlpromoter (SEQ ID NO: 16), a tkl1 promoter (SEQ ID NO: 17), a PID104295promoter (SEQ ID NO: 18), a dld1 promoter (SEQ ID NO: 19), a xyn4promoter (SEQ ID NO: 20), a PID72526 promoter (SEQ ID NO: 21), an axe1promoter (SEQ ID NO: 22), a hxk1 promoter (SEQ ID NO: 23), a dic1promoter (SEQ ID NO: 24), an opt promoter (SEQ ID NO: 25), a gut1promoter (SEQ ID NO: 26) and a pki1 promoter (SEQ ID NO: 27).

In related embodiments of the method, the endogenous POI is alignocellulose degrading enzyme. In certain embodiments, thelignocellulose degrading enzyme is selected from the group consisting ofcellulase enzymes, hemi-cellulase enzymes, or a combination thereof. Incertain embodiments, the cellulase enzymes are selected from the groupconsisting of cbh1, cbh2, egl1, egl2, egl3, egl4, egl5, egl6, bgl1,bgl2, swo1, cip1 and cip2. In another embodiment, the hemi-cellulaseenzymes are selected from the group consisting of xyn1, xyn2, xyn3,xyn4, bxl1, abf1, abf2, abf3, axe1, axe2, axe3, man1, agl1, agl2, agl3and gir1.

In other embodiments of the method, step (i) further comprises anintroduced polynucleotide construct encoding a heterologous POI. Incertain embodiments, the polynucleotide construct encoding theheterologous POI is expressed under the control of a cellulose-induciblegene promoter. In certain embodiments, a cellulose-inducible genepromoter is selected from cbh1, cbh2, egl1, egl2, xyn2 or stp1.

In other embodiments, the method further comprising a geneticmodification which expresses a polynucleotide encoding a wild-typexylanase regulator 1 (Xyr1) protein of SEQ ID NO: 48 or a variantxylanase regulator 1 (Xyr1) protein of SEQ ID NO: 46. In otherembodiments, the method further comprising a genetic modification whichreduces or prevents the expression of a gene encoding an endogenouscarbon catabolite repressor 1 (Cre1) protein or an Ace1 protein. In yetanother embodiment, the method further comprising a genetic modificationwhich comprises expressing an Ace2 protein.

In other embodiments, the disclosure is related to a method forproducing an endogenous protein of interest in a Trichoderma sp. fungalcell in the absence of an inducing substrate, the method comprising (i)introducing into the fungal cell a polynucleotide construct comprisingin the 5′ to 3′ direction: (a) a first nucleic acid sequence comprisinga promoter and (b) a second nucleic acid sequence operably linked to thefirst nucleic acid sequence, wherein the second nucleic acid sequenceencodes an Ace3 protein comprises about 90% sequence identity to SEQ IDNO: 12, and (ii) fermenting the cells of step (i) under conditionssuitable for fungal cell growth and protein production, wherein suchsuitable growth conditions do not include an inducing substrate. Inother embodiments, the polynucleotide construct comprises a thirdnucleic acid sequence 3′ and operably linked to the second nucleic acid,wherein the third nucleic acid sequence comprises a native ace3terminator sequence. In other embodiments of the method, thepolynucleotide construct is integrated into the fungal cell genome. Incertain embodiments, the polynucleotide construct is integrated into atelomere site of the fungal cell genome. In certain other embodiments,the polynucleotide construct is integrated into a glucoamylase (gla1)gene locus of the fungal cell genome. The other embodiment of themethod, the polynucleotide construct comprises a nucleotide sequencecomprising about 90% sequence identity to SEQ ID NO: 4, SEQ ID NO: 11 orSEQ ID NO: 13. In certain embodiments, the encoded Ace3 proteincomprises an amino acid sequence comprising 95% sequence identity to SEQID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 14. In certain other embodiments,the encoded Ace3 protein comprises an amino acid sequence of SEQ ID NO:6, SEQ ID NO: 12 or SEQ ID NO: 14.

Thus, in other embodiments of the method 8, the endogenous POI is alignocellulose degrading enzyme. In particular embodiments, thelignocellulose degrading enzyme is selected from the group consisting ofcellulase enzymes, hemi-cellulase enzymes, or a combination thereof. Inanother embodiment, the cellulase enzymes are selected from the groupconsisting of cbh1, cbh2, egl1, egl2, egl3, egl4, egl5, egl6, bgl1,bgl2, swo1, cip1 and cip2. In other embodiments, the hemi-cellulaseenzymes are selected from the group consisting of xyn1, xyn2, xyn3,xyn4, bxl1, abf1, abf2, abf3, axe1, axe2, axe3, man1, agl1, agl2, agl3and gir1.

In another embodiment of the method, step (i) further comprises anintroduced polynucleotide construct encoding a heterologous POI. Incertain embodiments, the polynucleotide construct encoding theheterologous POI is expressed under the control of a cellulose-induciblegene promoter. In certain other embodiments of the method, the step (i)promoter is selected from the group consisting of a rev3 promoter (SEQID NO: 15), a bxl promoter (SEQ ID NO: 16), a tkl1 promoter (SEQ ID NO:17, a PID104295 promoter (SEQ ID NO: 18), a dld1 promoter (SEQ ID NO:19), a xyn4 promoter (SEQ ID NO: 20), a PID72526 promoter (SEQ ID NO:21), an axe1 promoter (SEQ ID NO: 22), a hxk1 promoter (SEQ ID NO: 23),a dic1 promoter (SEQ ID NO: 24), an opt promoter (SEQ ID NO: 25), a gut1promoter (SEQ ID NO: 26) and a pki1 promoter (SEQ ID NO: 27). In anotherembodiment, the method further comprises a genetic modification whichexpresses a polynucleotide encoding a wild-type xylanase regulator 1(Xyr1) protein of SEQ ID NO: 48 or a variant xylanase regulator 1 (Xyr1)protein of SEQ ID NO: 46. In certain other embodiments, the methodfurther comprises a genetic modification which reduces or prevents theexpression of a gene encoding an endogenous carbon catabolite repressor1 (Cre1) protein or an Ace1 protein. In yet other embodiments, themethod further comprises a genetic modification which comprisesexpressing an Ace2 protein.

In certain other embodiments, the disclosure is directed to a method forproducing a heterologous protein of interest in a Trichoderma sp. fungalcell in the absence of an inducing substrate, the method comprising (i)introducing into the fungal cell a polynucleotide construct comprisingin the 5′ to 3′ direction: (a) a first nucleic acid sequence comprisinga constitutive promoter and (b) a second nucleic acid sequence operablylinked to the first nucleic acid sequence, wherein the second nucleicacid sequence encodes an Ace3 protein comprising about 90% sequenceidentity to SEQ ID NO: 6 and comprises “Lys-Ala-Ser-Asp” as the lastfour C-terminal amino acids, and (ii) fermenting the cells of step (i)under conditions suitable for fungal cell growth and protein production,wherein such suitable growth conditions do not include an inducingsubstrate. Thus, in certain embodiments of the method, the fungal cellcomprises an introduced polynucleotide construct encoding a heterologousPOI, wherein the construct is introduced into in the fungal cell priorto step (i), during step (i) or after step (i). In another embodiment,the polynucleotide construct comprises a third nucleic acid sequence 3′and operably linked to the second nucleic acid, wherein the thirdnucleic acid sequence comprises a native ace3 terminator sequence. Inother embodiments of the method, the polynucleotide construct isintegrated into the fungal cell genome. In certain embodiments, thepolynucleotide construct is integrated into a telomere site of thefungal cell genome. In other embodiments, the polynucleotide constructis integrated into a glucoamylase (gla1) gene locus of the fungal cellgenome.

In certain other embodiments of the method, the polynucleotide constructcomprises a nucleotide sequence comprising about 90% sequence identityto SEQ ID NO: 4, SEQ ID NO: 11 or SEQ ID NO: 13. In another embodiment,the encoded Ace3 protein comprises an amino acid sequence comprising 95%sequence identity to SEQ ID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 14. Inother embodiments, the encoded Ace3 protein comprises an amino acidsequence of SEQ ID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 14. In otherembodiments, the polynucleotide construct encoding the heterologous POIis expressed under the control of a cellulose-inducible gene promoter.In certain other embodiments, the heterologous POI is selected from thegroup consisting of an α-amylase, an alkaline α-amylase, a β-amylase, acellulase, a dextranase, an α-glucosidase, an α-galactosidase, aglucoamylase, a hemicellulase, a pentosanase, a xylanase, an invertase,a lactase, a naringanase, a pectinase, a pullulanase, an acid protease,an alkali protease, a bromelain, a neutral protease, a papain, a pepsin,a peptidase, a rennet, a rennin, a chymosin, a subtilisin, athermolysin, an aspartic proteinase, a trypsin, a lipase, an esterase, aphospholipase, a phosphatase, a phytase, an amidase, an iminoacylase, aglutaminase, a lysozyme, a penicillin acylase; an isomerase, anoxidoreductases, a catalase, a chloroperoxidase, a glucose oxidase, ahydroxysteroid dehydrogenase, a peroxidase, a lyase, an asparticβ-decarboxylase, a fumarase, a histadase, a transferase, a ligase, anaminopeptidase, a carboxypeptidase, a chitinase, a cutinase, adeoxyribonuclease, an α-galactosidase, a β-galactosidase, aβ-glucosidase, a laccase, a mannosidase, a mutanase, a polyphenoloxidase, a ribonuclease and a transglutaminase.

In other embodiments of the method, the step (i) promoter is selectedfrom the group consisting of a rev3 promoter (SEQ ID NO: 15), a bxlpromoter (SEQ ID NO: 16), a tkl1 promoter (SEQ ID NO: 17, a PID104295promoter (SEQ ID NO: 18), a dld1 promoter (SEQ ID NO: 19), a xyn4promoter (SEQ ID NO: 20), a PID72526 promoter (SEQ ID NO: 21), an axe1promoter (SEQ ID NO: 22), a hxk1 promoter (SEQ ID NO: 23), a dic1promoter (SEQ ID NO: 24), an opt promoter (SEQ ID NO: 25), a gut1promoter (SEQ ID NO: 26) and a pki1 promoter (SEQ ID NO: 27). In yetother embodiments, the method further comprises a genetic modificationwhich expresses a polynucleotide encoding a wild-type xylanase regulator1 (Xyr1) protein of SEQ ID NO: 48 or a variant xylanase regulator 1(Xyr1) protein of SEQ ID NO: 46. In another embodiment, the methodfurther comprises a genetic modification which reduces or prevents theexpression of a gene encoding an endogenous carbon catabolite repressor1 (Cre1) protein or an Ace1 protein. In yet other embodiments, themethod further comprises a genetic modification which comprisesexpressing an Ace2 protein.

In another embodiment, the disclosure is directed to a method forproducing a heterologous protein of interest in a Trichoderma sp. fungalcell in the absence of an inducing substrate, the method comprising (i)introducing into the fungal cell a polynucleotide construct comprisingin the 5′ to 3′ direction: (a) a first nucleic acid sequence comprisinga constitutive promoter and (b) a second nucleic acid sequence operablylinked to the first nucleic acid sequence, wherein the second nucleicacid sequence encodes an Ace3 protein comprises about 90% sequenceidentity to SEQ ID NO: 12, and (ii) fermenting the cells of step (i)under conditions suitable for fungal cell growth and protein production,wherein such suitable growth conditions do not include an inducingsubstrate. In particular embodiments, the fungal cell comprises anintroduced polynucleotide construct encoding a heterologous POI, whereinthe construct is introduced into in the fungal cell prior to step (i),during step (i) or after step (i). In other embodiments, thepolynucleotide construct comprises a third nucleic acid sequence 3′ andoperably linked to the second nucleic acid, wherein the third nucleicacid sequence comprises a native ace3 terminator sequence. In otherembodiments of the method, the polynucleotide construct is integratedinto the fungal cell genome. In certain embodiments, the polynucleotideconstruct is integrated into a telomere site of the fungal cell genome.In other embodiments, the polynucleotide construct is integrated into aglucoamylase (gla1) gene locus of the fungal cell genome. In yet otherembodiments, the polynucleotide construct comprises a nucleotidesequence comprising about 90% sequence identity to SEQ ID NO: 4, SEQ IDNO: 11 or SEQ ID NO: 13. In certain embodiments, encoded Ace3 proteincomprises an amino acid sequence comprising 95% sequence identity to SEQID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 14. In another embodiment, theencoded Ace3 protein comprises an amino acid sequence of SEQ ID NO: 6,SEQ ID NO: 12 or SEQ ID NO: 14. In another embodiment of the method, thepolynucleotide construct encoding the heterologous POI is expressedunder the control of a cellulose-inducible gene promoter. In certainembodiments, the heterologous POI is selected from the group consistingof an α-amylase, an alkaline α-amylase, a β-amylase, a cellulase, adextranase, an α-glucosidase, an α-galactosidase, a glucoamylase, ahemicellulase, a pentosanase, a xylanase, an invertase, a lactase, anaringanase, a pectinase, a pullulanase, an acid protease, an alkaliprotease, a bromelain, a neutral protease, a papain, a pepsin, apeptidase, a rennet, a rennin, a chymosin, a subtilisin, a thermolysin,an aspartic proteinase, a trypsin, a lipase, an esterase, aphospholipase, a phosphatase, a phytase, an amidase, an iminoacylase, aglutaminase, a lysozyme, a penicillin acylase; an isomerase, anoxidoreductases, a catalase, a chloroperoxidase, a glucose oxidase, ahydroxysteroid dehydrogenase, a peroxidase, a lyase, an asparticβ-decarboxylase, a fumarase, a histadase, a transferase, a ligase, anaminopeptidase, a carboxypeptidase, a chitinase, a cutinase, adeoxyribonuclease, an α-galactosidase, a β-galactosidase, aβ-glucosidase, a laccase, a mannosidase, a mutanase, a polyphenoloxidase, a ribonuclease and a transglutaminase.

In other embodiments of the method, the step (i) promoter is selectedfrom the group consisting of a rev3 promoter (SEQ ID NO: 15), a bxlpromoter (SEQ ID NO: 16), a tkl1 promoter (SEQ ID NO: 17, a PID104295promoter (SEQ ID NO: 18), a dld1 promoter (SEQ ID NO: 19), a xyn4promoter (SEQ ID NO: 20), a PID72526 promoter (SEQ ID NO: 21), an axe1promoter (SEQ ID NO: 22), a hxk1 promoter (SEQ ID NO: 23), a dic1promoter (SEQ ID NO: 24), an opt promoter (SEQ ID NO: 25), a gut1promoter (SEQ ID NO: 26) and a pki1 promoter (SEQ ID NO: 27). In yetother embodiments, the method further comprises a genetic modificationwhich expresses a polynucleotide encoding a wild-type xylanase regulator1 (Xyr1) protein of SEQ ID NO: 48 or a variant xylanase regulator 1(Xyr1) protein of SEQ ID NO: 46. In another embodiment, the methodfurther comprises a genetic modification which reduces or prevents theexpression of a gene encoding an endogenous carbon catabolite repressor1 (Cre1) protein or an Ace1 protein. In yet other embodiments, themethod further comprises a genetic modification which comprisesexpressing an Ace2 protein.

In certain other embodiments, the disclosure is related to a method forgenetically modifying a Trichoderma reesei strain for increasedproduction of an endogenous protein in the absence of an inducingsubstrate, the method comprising (i) screening and identifying a T.reesei strain comprising a genomic copy of an ace3 gene encoding anAce3-S protein of SEQ ID NO: 3, an Ace3-SC protein of SEQ ID NO: 8 or anAce3-LC protein of SEQ ID NO: 10, wherein the T. reesei strainidentified does not comprise a genomic copy of an ace3 gene encoding anAce3-L protein of SEQ ID NO: 6, an Ace3-LN protein of SEQ ID NO: 14 oran Ace3-EL protein of SEQ ID NO: 12, (ii) introducing into the T. reeseistrain identified in step (i) a polynucleotide construct comprising inthe 5′ to 3′ direction: (a) a first nucleic acid sequence comprising apromoter and (b) a second nucleic acid sequence operably linked to thefirst nucleic acid sequence, wherein the second nucleic acid sequenceencodes an Ace3 protein comprising about 90% sequence identity to SEQ IDNO: 6 and comprises “Lys-Ala-Ser-Asp” as the last four C-terminal aminoacids or the second nucleic acid sequence encodes an Ace-3 proteincomprising about 90% sequence identity to SEQ ID NO: 12, and (iii)fermenting the cells of step (ii) under conditions suitable for fungalcell growth and protein production, wherein such suitable growthconditions do not include an inducing substrate.

In certain other embodiments, the disclosure is directed to a variantfilamentous fungal cell derived from a parental filamentous fungal cell,the variant cell comprising a native ace3 gene promoter replaced by analternative promoter, wherein the variant cell produces an increasedamount of a protein of interest (POI) in the absence of an inducingsubstrate relative to the parental cell, wherein the variant andparental cells are cultivated under similar conditions. In particularembodiments, the alternative promoter is a Trichoderma reesei promoter.In another embodiment, the alternative promoter is a promoter selectedfrom the group consisting of a rev3 promoter (SEQ ID NO: 15), a bxlpromoter (SEQ ID NO: 16), a tkl1 promoter (SEQ ID NO: 17, a PID104295promoter (SEQ ID NO: 18), a dld1 promoter (SEQ ID NO: 19), a xyn4promoter (SEQ ID NO: 20), a PID72526 promoter (SEQ ID NO: 21), an axe1promoter (SEQ ID NO: 22), a hxk1 promoter (SEQ ID NO: 23), a dic1promoter (SEQ ID NO: 24), an opt promoter (SEQ ID NO: 25), a gut1promoter (SEQ ID NO: 26) and a pki1 promoter (SEQ ID NO: 27).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a schematic representation of the Ace3 protein codingregions. More particularly, FIG. 1A presents a schematic representationof the Ace3 protein coding regions based on the annotation of T. reeseistrains QM6a and RUT-C30, which are aligned (FIG. 1A) to the same DNAsequence in the genome. The predicted exons and introns are shown asarrows and dash lines, respectively. The dashed-vertical line indicatesa non-sense mutation in RUT-C30 genome. The cloned ace3-S (short) openreading frame of SEQ ID NO: 2 and the cloned ace3-L (long) open readingframe of SEQ ID NO: 5 were screened and tested as set forth in theinstant application. Set forth in FIG. 1B-FIG. 1D is an amino acidalignment of the Ace3-L protein (SEQ ID NO: 6) from T. reesei strainRUT-C30, the Ace3-S protein from T. reesei strain QM6a (SEQ ID NO: 3),the Ace3-SC protein (SEQ ID NO: 8), the Ace3-LN protein (SEQ ID NO: 14),the Ace3-LC protein (SEQ ID NO: 10) and the Ace3-EL protein (SEQ ID NO:12).

FIG. 2 shows a schematic representation of the Ace3-expression vectorspYL1 (FIG. 2A), pYL2 (FIG. 2B), pYL3 (FIG. 2C) and pYL4 (FIG. 2D).

FIG. 3 presents a Polyacrylamide Gel Electrophoresis (SDS-PAGE) of T.reesei parental and variant cell supernatants, wherein the parental andvariant cells were grown in defined medium in srMTP containing 20%lactose (lac) or 20% glucose (glu). Equal volumes of culturesupernatants were loaded in each lane. M is molecular weight marker andthe parental T. reesei strain served as a control strain.

FIG. 4 presents a SDS-PAGE of T. reesei parental and variant cellsupernatants, wherein the parental and variant cells were grown indefined medium supplemented with either 1.5% glucose/sophorose (sop) or1.5% glucose (glu) in shake flasks. Equal volumes of culture supernatantwere loaded in each lane. The total protein concentrations of theculture supernatants are listed at the bottom of each correspondinglane. M is molecular weight marker and KD is kilodalton.

FIG. 5 presents a SDS-PAGE of T. reesei parental and variant cells grownin defined medium with either glucose/sophorose (sop) or glucose (glu)as carbon sources in 2 L fermenters. M is molecular weight marker andzero (0) is seed culture supernatant.

FIG. 6 presents a schematic diagram of a promoter replacement constructmade by fusing a DNA fragment comprising a 5′ region upstream of thenative promoter at the ace3 locus, a loxP-flanked hygromycinB-resistance (selectable marker) cassette and a DNA fragment comprisinga promoter of interest operably fused (linked) to the 5′ end of the ace3ORF.

FIG. 7 shows a schematic representation of Ace3-expression vector pYL8comprising the dic1 promoter.

FIG. 8 shows a SDS-PAGE of T. reesei parental and its modified(daughter) cell supernatants, wherein the parental and modified strainswere grown in defined medium supplemented with either 2.5%glucose/sophorose (“Sop”, inducing condition) or 2.5% glucose (“Glu”,non-inducing condition) in shake flasks. Equal volumes of culturesupernatant were loaded in each lane. M is molecular weight marker andKD is kilodalton.

FIG. 9 shows protein production in small scale (2 L) fermentation. TheT. reesei parental strain and daughter strain “LT83” were grown indefined medium with either glucose/sophorose (Sop, inducing condition)or glucose (Glu, non-inducing condition) as carbon sources. The totalprotein produced by the parental strain on glucose/sophorose at the endof fermentation was arbitrarily set at 100, and the relative amounts ofprotein produced by each strain at each time points were plotted.

FIG. 10 shows a SDS-PAGE of T. reesei parental and its modified(daughter) cell supernatants, wherein the parental and modified strainswere grown in defined medium supplemented with either 2.5%glucose/sophorose (“Sop”, inducing condition) or 2.5% glucose (“Glu”,non-inducing condition) in shake flasks. Equal volumes of culturesupernatant were loaded in each lane. M is molecular weight marker andKD is kilodalton.

FIG. 11 presents a schematic image of the ace3 locus. The arrows at the5′-end (N-terminus) of the ace3 locus indicate the differenttranscription start sites in the form suggested by cDNA sequence (arrow1), the RutC-30 annotated form (arrow 2) and QM6a annotated form (arrow3). The arrows at the 3′-end (C-terminus) of the ace3 locus indicate thedifferent Stop codons in the RutC-30 annotated form (arrow 4) and QM6aannotated form (arrow 5).

FIG. 12 shows a schematic image of the different ace3 forms cloned.Thus, as presented in FIG. 12 and described in Example 6, the followingace3 forms were cloned and tested: “ace3-S” of SEQ ID NO: 1, comprisinga 1,713 bp Exon 3, a 148 bp Intron 3 and a 177 bp Exon 4, “ace3-SC” ofSEQ ID NO: 7, comprising a 1,713 bp Exon 3, a 148 bp Intron 3 and a 144bp Exon 4, “ace3-L” of SEQ ID NO: 4, comprising a 258 bp Exon 2, a 423bp Intron 2, a 1,635 bp Exon 3, a 148 bp Intron 3 and a 144 bp Exon 4,“ace3-LC” of SEQ ID NO: 9, comprising a 258 bp Exon 2, a 423 bp Intron2, a 1,635 bp Exon 3, a 148 bp Intron 3 and a 177 bp Exon 4, “ace3-EL”of SEQ ID NO: 11, comprising a 61 bp Exon 1, a 142 bp Intron 1, a 332 bpExon 2, a 423 bp Intron 2, a 1,635 bp Exon 3, a 148 bp Intron 3 and a144 bp Exon 4, and “ace3-LN” of SEQ ID NO: 13, comprising a 258 bp Exon2, a 1,635 bp Exon 3, a 148 bp Intron 3 and a 144 bp Exon 4.

FIG. 13 shows the nucleic acid sequence of the ace3-SC gene form (SEQ IDNO: 7; comprising a 1,713 bp Exon 3, a 148 bp Intron 3 and a 144 bp Exon4) and the encoded Ace3-SC protein sequence (SEQ ID NO: 8). As presentedin FIG. 13 for the ace3-SC gene form, nucleotides shown in bold blacktext represent intron sequences.

FIG. 14 shows the nucleic acid sequence of the ace3-S gene form (SEQ IDNO: 1; comprising a 1,713 bp Exon 3, a 148 bp Intron 3 and a 177 bp Exon4) and the encoded Ace3-S protein sequence (SEQ ID NO: 3). As presentedin FIG. 14 for the ace3-S gene form, nucleotides shown in bold blacktext represent intron sequences.

FIG. 15 shows the nucleic acid sequence of the ace3-L gene form (SEQ IDNO: 4, comprising a 258 bp Exon 2, a 423 bp Intron 2, a 1,635 bp Exon 3,a 148 bp Intron 3 and a 144 bp Exon 4) and the encoded Ace3-L proteinsequence (SEQ ID NO: 6). As presented in FIG. 15 for the ace3-L geneform, nucleotides shown in bold black text represent intron sequences.

FIG. 16 shows the nucleic acid sequence of the ace3-LC gene form (SEQ IDNO: 9, comprising a 258 bp Exon 2, a 423 bp Intron 2, a 1,635 bp Exon 3,a 148 bp Intron 3 and a 177 bp Exon 4) and the encoded Ace3-LC proteinsequence (SEQ ID NO: 10). As presented in FIG. 16 for the ace3-LC geneform, nucleotides shown in bold black text represent intron sequences.

FIG. 17 shows the nucleic acid sequence of the ace3-EL gene form (SEQ IDNO: 11, comprising a 61 bp Exon 1, a 142 bp Intron 1, a 332 bp Exon 2, a423 bp Intron 2, a 1,635 bp Exon 3, a 148 bp Intron 3 and a 144 bp Exon4) and the encoded Ace3-EL protein sequence (SEQ ID NO: 12). Aspresented in FIG. 17 for the ace3-EL gene form, nucleotides shown inbold black text represent intron sequences.

FIG. 18 shows the nucleic acid sequence of the ace3-LN gene form (SEQ IDNO: 13, comprising a 258 bp Exon 2, a 1,635 bp Exon 3, a 148 bp Intron 3and a 144 bp Exon 4) and the encoded Ace3-LN protein sequence (SEQ IDNO: 14). As presented in FIG. 18 for the ace3-LN gene form, nucleotidesshown in bold black text represent intron sequences.

FIG. 19 presents a schematic diagram of the arrangement of DNA fragmentsdesigned for integration of ace3 forms at the gla1 locus.

FIG. 20 shows a schematic representation of vector pMCM3282.

FIG. 21 shows a schematic representation of vector pCHL760.

FIG. 22 shows a schematic representation of vector pCHL761.

FIG. 23 shows a SDS-PAGE of secreted proteins produced by submergedcultures (i.e., shake flasks) of T. reesei parental cells (FIG. 23, cellID 1275.8.1) and variant T. reesei (daughter) cells (FIG. 23, cell IDNos. 2218, 2219, 2220, 2222 and 2223) under inducing (“Glu/Sop”) andnon-inducing (“Glu”) culture conditions.

BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

The following sequences comply with 37 C.F.R. §§ 1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequenceand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with WIP Standard ST.25 (2009) and the sequence listingrequirements of the European Patent Convention (EPC) and the PatentCooperation Treaty (PCT) rules 5.2 and 49.5(a-bis), and section 208 andAnnex C of the administrative instructions. The symbols and format usedfor nucleotide and amino acid sequence data comply with the rules setforth in 37 C.F.R. § 1.822.

SEQ ID NO: 1 is a Trichoderma reesei wild-type strain QM6a nucleic acidsequence comprising a gene encoding an Ace3-S protein of SEQ ID NO: 3.

SEQ ID NO: 2 is a nucleic acid sequence open reading frame (ORF)encoding an Ace3-S protein of SEQ ID NO: 3.

SEQ ID NO: 3 is the amino acid sequence of a Trichoderma reesei (strainQM6a) Ace3 protein, designated hereinafter, “Ace3-S”.

SEQ ID NO: 4 is a Trichoderma reesei strain Rut-C30 nucleic acidsequence comprising a gene encoding an Ace3-L protein of SEQ ID NO: 6.

SEQ ID NO: 5 is a nucleic acid sequence ORF encoding an Ace3-L proteinof SEQ ID NO: 6.

SEQ ID NO: 6 is the amino acid sequence of a Trichoderma reesei (strainRut-C30) Ace3 protein, designated hereinafter, “Ace3-L”.

SEQ ID NO: 7 is a Trichoderma reesei nucleic acid sequence comprising agene encoding an Ace3-SC protein of SEQ ID NO: 8.

SEQ ID NO: 8 is the amino acid sequence of a Trichoderma reesei Ace3protein, designated hereinafter, “Ace3-SC”.

SEQ ID NO: 9 is a Trichoderma reesei nucleic acid sequence comprising agene encoding an Ace3-LC protein of SEQ ID NO: 10.

SEQ ID NO: 10 is the amino acid sequence of a Trichoderma reesei Ace3protein, designated hereinafter, “Ace3-LC”.

SEQ ID NO: 11 is a Trichoderma reesei nucleic acid sequence comprising agene encoding an Ace3-EL protein of SEQ ID NO: 12.

SEQ ID NO: 12 is the amino acid sequence of a Trichoderma reesei Ace3protein, designated hereinafter, “Ace3-EL”.

SEQ ID NO: 13 is a Trichoderma reesei nucleic acid sequence comprising agene encoding an Ace3-LN protein of SEQ ID NO: 14.

SEQ ID NO: 14 is the amino acid sequence of a Trichoderma reesei Ace3protein, designated hereinafter, “Ace3-LN”.

SEQ ID NO: 15 is a nucleic acid sequence comprising a rev3 promotersequence.

SEQ ID NO: 16 is a nucleic acid sequence comprising a β-xyl promotersequence.

SEQ ID NO: 17 is a nucleic acid sequence comprising a tki1 promotersequence.

SEQ ID NO: 18 is a nucleic acid sequence comprising a PID104295 promotersequence.

SEQ ID NO: 19 is a nucleic acid sequence comprising a dld1 promotersequence.

SEQ ID NO: 20 is a nucleic acid sequence comprising a xyn4 promotersequence.

SEQ ID NO: 21 is a nucleic acid sequence comprising a PID72526 promotersequence.

SEQ ID NO: 22 is a nucleic acid sequence comprising an axe3 promotersequence.

SEQ ID NO: 23 is a nucleic acid sequence comprising a hxk1 promotersequence.

SEQ ID NO: 24 is a nucleic acid sequence comprising a dic1 promotersequence.

SEQ ID NO: 25 is a nucleic acid sequence comprising an opt promotersequence.

SEQ ID NO: 26 is a nucleic acid sequence comprising a gut1 promotersequence.

SEQ ID NO: 27 is a nucleic acid sequence comprising a pki1 promotersequence.

SEQ ID NO: 28 is a nucleic acid sequence of primer TP13.

SEQ ID NO: 29 is a nucleic acid sequence of primer TP14.

SEQ ID NO: 30 is a nucleic acid sequence of primer TP15.

SEQ ID NO: 31 is a nucleic acid sequence of primer TP16.

SEQ ID NO: 32 is a nucleic acid sequence of primer TP17.

SEQ ID NO: 33 is a nucleic acid sequence of primer TP18.

SEQ ID NO: 34 is a nucleic acid sequence of primer TP19.

SEQ ID NO: 35 is a nucleic acid sequence of primer TP20.

SEQ ID NO: 36 is a nucleic acid sequence of primer TP21.

SEQ ID NO: 37 is a nucleic acid sequence of primer TP22.

SEQ ID NO: 38 is a nucleic acid sequence of primer TP23.

SEQ ID NO: 39 is a nucleic acid sequence of primer TP24.

SEQ ID NO: 40 is a nucleic acid sequence of primer TP25.

SEQ ID NO: 41 is a nucleic acid sequence of primer TP26.

SEQ ID NO: 42 is a nucleic acid sequence of plasmid pYL1.

SEQ ID NO: 43 is a nucleic acid sequence of plasmid pYL2.

SEQ ID NO: 44 is a nucleic acid sequence of plasmid pYL3.

SEQ ID NO: 45 is a nucleic acid sequence of plasmid pYL4.

SEQ ID NO: 46 is an amino acid sequence of a T. reesei xyr1 (A824V)variant protein.

SEQ ID NO: 47 is an amino acid sequence of a T. reesei Ace2 protein.

SEQ ID NO: 48 is an amino acid sequence of a T. reesei wild-type xyr1protein.

SEQ ID NO: 49 is primer nucleic acid sequence.

SEQ ID NO: 50 is primer nucleic acid sequence.

SEQ ID NO: 51 is primer nucleic acid sequence.

SEQ ID NO: 52 is primer nucleic acid sequence.

SEQ ID NO: 53 is primer nucleic acid sequence.

SEQ ID NO: 54 is primer nucleic acid sequence.

SEQ ID NO: 55 is primer nucleic acid sequence.

SEQ ID NO: 56 is primer nucleic acid sequence.

SEQ ID NO: 57 is primer nucleic acid sequence.

SEQ ID NO: 58 is primer nucleic acid sequence.

SEQ ID NO: 59 is primer nucleic acid sequence.

SEQ ID NO: 60 is primer nucleic acid sequence.

SEQ ID NO: 61 is primer nucleic acid sequence.

SEQ ID NO: 62 is primer nucleic acid sequence.

SEQ ID NO: 63 is primer nucleic acid sequence.

SEQ ID NO: 64 is primer nucleic acid sequence.

SEQ ID NO: 65 is primer nucleic acid sequence.

SEQ ID NO: 66 is primer nucleic acid sequence.

SEQ ID NO: 67 is primer nucleic acid sequence.

SEQ ID NO: 68 is primer nucleic acid sequence.

SEQ ID NO: 69 is primer nucleic acid sequence.

SEQ ID NO: 70 is primer nucleic acid sequence.

SEQ ID NO: 71 is primer nucleic acid sequence.

SEQ ID NO: 72 is primer nucleic acid sequence.

SEQ ID NO: 73 is primer nucleic acid sequence.

SEQ ID NO: 74 is primer nucleic acid sequence.

SEQ ID NO: 75 is primer nucleic acid sequence.

SEQ ID NO: 76 is primer nucleic acid sequence.

SEQ ID NO: 77 is primer nucleic acid sequence.

SEQ ID NO: 78 is primer nucleic acid sequence.

SEQ ID NO: 79 is primer nucleic acid sequence.

SEQ ID NO: 80 is primer nucleic acid sequence.

SEQ ID NO: 81 is primer nucleic acid sequence.

SEQ ID NO: 82 is an artificial nucleic acid sequence.

SEQ ID NO: 83 is an artificial nucleic acid sequence.

SEQ ID NO: 84 is an artificial nucleic acid sequence.

SEQ ID NO: 85 is an artificial nucleic acid sequence.

SEQ ID NO: 86 is an artificial nucleic acid sequence.

SEQ ID NO: 87 is an artificial nucleic acid sequence.

SEQ ID NO: 88 is an artificial nucleic acid sequence.

SEQ ID NO: 89 is an artificial nucleic acid sequence.

SEQ ID NO: 90 is an artificial nucleic acid sequence.

SEQ ID NO: 91 is an artificial nucleic acid sequence.

SEQ ID NO: 92 is primer nucleic acid sequence.

SEQ ID NO: 93 is primer nucleic acid sequence.

SEQ ID NO: 94 is primer nucleic acid sequence.

SEQ ID NO: 95 is primer nucleic acid sequence.

SEQ ID NO: 96 is primer nucleic acid sequence.

SEQ ID NO: 97 is primer nucleic acid sequence.

SEQ ID NO: 98 is an amino acid sequence comprising a forty-five (45)amino acid fragment of N-terminal sequence of the Ace3-EL protein of SEQID NO: 12.

SEQ ID NO: 99 is a nucleic acid sequence ORF encoding an Ace3-SCprotein.

SEQ ID NO: 100 is a nucleic acid sequence ORF encoding an Ace3-LCprotein.

SEQ ID NO: 101 is a nucleic acid sequence ORF encoding an Ace3-ELprotein.

SEQ ID NO: 102 is a nucleic acid sequence ORF encoding an Ace3-LNprotein.

DETAILED DESCRIPTION I. Overview

Certain embodiments of the disclosure are directed to variantfilamentous fungal cells, compositions thereof and methods thereof forincreased production of one or more proteins of interest. Moreparticularly, certain embodiments of the disclosure are directed tovariant filamentous fungal cells capable of producing one or moreproteins of interest in the absence of an inducing feed (i.e., aninducing substrate such as lactose, sophorose, gentiobiose, celluloseand the like). Thus, certain embodiments of the disclosure are directedto variant filamentous fungal (host) cells derived from parentalfilamentous fungal cells, wherein the variant host cells comprise agenetic modification which enables the expression of a gene of interest(encoding a protein of interest) in the absence of inducing substrate.The gene of interest (encoding a protein of interest) can be anendogenous filamentous fungal cell gene (e.g., cbh1, chb2, xyn1, xyn2,xyn3, egl1, egl2, egl3, bgl1, bgl2, and the like) or a gene heterologousto the filamentous fungal cell.

Thus, in certain other embodiments, a variant fungal host cell of thedisclosure comprises a genetic modification which increases theexpression of an “activator of cellulase expression 3” (ace3) geneencoding an Ace3 protein selected from the group consisting of an Ace3-Lprotein (SEQ ID NO: 6), an Ace3-EL protein (SEQ ID NO: 12) and anAce3-LN protein (SEQ ID NO: 14). In other embodiments, a variant fungalhost cell of the disclosure comprises a genetic modification whichincreases the expression of a polynucleotide open reading frame (ORF)encoding an Ace3 protein selected from the group consisting of an Ace3-Lprotein (SEQ ID NO: 6), an Ace3-EL protein (SEQ ID NO: 12) and anAce3-LN protein (SEQ ID NO: 14). Thus, in certain embodiments, a variantfungal host cell of the disclosure comprises a genetic modificationwhich increases the expression/production of an Ace3 gene or ORF thereofencoding a Ace3 protein comprising about 90% to about 99% sequenceidentity to an Ace3 protein selected from the group consisting of anAce3-L protein (SEQ ID NO: 6), an Ace3-EL protein (SEQ ID NO: 12) and anAce3-LN protein (SEQ ID NO: 14).

In certain embodiments, the genetic modification which increases theexpression of an Ace3 protein (i.e., an Ace3-L, Ace3-EL or Ace3-LNprotein) is an ace3 expression cassette which has been integrated intothe genome (chromosome) of the filamentous fungal host cell. In otherembodiments, the genetic modification which increases expression of anAce3 protein in a filamentous fungal cell is an episomally maintainedplasmid construct comprising an ace3 expression cassette (i.e., encodingan Ace3-L, Ace3-EL or Ace3-LN protein). In other embodiments, thegenetic modification which increases the expression of an ace3 geneencoding an Ace3-L, Ace3-EL or Ace3-LN protein in a filamentous fungalcell is a telomeric vector/plasmid integrated in a telomere site. Incertain embodiments, such expression cassettes or plasmids are presentin more than one copy. In certain other embodiments, the ace3 gene, orace3 ORF, is operably linked to a heterologous promoter. In otherembodiments, the genetic modification which increases the expression anace3 gene (or ORF thereof) encoding an Ace3-L, Ace3-EL or Ace3-LNprotein in a filamentous fungal cell is a modification of the nativeace3 promoter (i.e., the ace3 promoter region natively associated withthe ace3 gene) which modification alters the expression of an ace3 geneencoding an Ace3-L, Ace3-EL or Ace3-LN protein.

II. Definitions

Prior to describing the present compositions and methods in furtherdetail, the following terms and phrases are defined. Terms not definedshould be accorded their ordinary meaning as used and known to oneskilled in the art.

All publications and patents cited in this specification are hereinincorporated by reference.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the present compositions andmethods. The upper and lower limits of these smaller ranges mayindependently be included in the smaller ranges and are also encompassedwithin the present compositions and methods, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the present compositions and methods.

Certain ranges are presented herein with numerical values being precededby the term “about”. The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating un-recited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number. For example,in connection with a numerical value, the term “about” refers to a rangeof ⁻10% to ⁺10% of the numerical value, unless the term is otherwisespecifically defined in context. In another example, the phrase a “pHvalue of about 6” refers to pH values of from 5.4 to 6.6, unless the pHvalue is specifically defined otherwise.

The headings provided herein are not limitations of the various aspectsor embodiments of the present compositions and methods which can be hadby reference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification as a whole.

In accordance with this Detailed Description, the followingabbreviations and definitions apply. Note that the singular forms “a,”“an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an enzyme” includesa plurality of such enzymes, and reference to “the dosage” includesreference to one or more dosages and equivalents thereof known to thoseskilled in the art, and so forth.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely”,“only”, “excluding”, “not including” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is further noted that the term “comprising”, as used herein, means“including, but not limited to”, the component(s) after the term“comprising”. The component(s) after the term “comprising” are requiredor mandatory, but the composition comprising the component(s) mayfurther include other non-mandatory or optional component(s).

It is also noted that the term “consisting of,” as used herein, means“including and limited to”, the component(s) after the term “consistingof”. The component(s) after the term “consisting of” are thereforerequired or mandatory, and no other component(s) are present in thecomposition.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentcompositions and methods described herein. Any recited method can becarried out in the order of events recited or in any other order whichis logically possible.

As used herein, the term “Ascomycete fungal cell” refers to any organismin the Division Ascomycota in the Kingdom Fungi. Examples of Ascomycetesfungal cells include, but are not limited to, filamentous fungi in thesubphylum Pezizomycotina, such as Trichoderma spp., Aspergillus spp.,Myceliophthora spp. and Penicillium spp.

As used herein, the term “filamentous fungus” refers to all filamentousforms of the subdivision Eumycota and Oomycota. For example, filamentousfungi include, without limitation, Acremonium, Aspergillus, Emericella,Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium,Scytalidium, Thielavia, Tolypocladium, or Trichoderma species. In someembodiments, the filamentous fungus may be an Aspergillus aculeatus,Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae.

In some embodiments, the filamentous fungus is a Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum. In some embodiments, the filamentous fungus is a Humicolainsolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Scytalidium thermophilum, or Thielavia terrestris.

In some embodiments, a filamentous fungus is a Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei orTrichoderma viride. In some embodiments, the filamentous fungus is aTrichoderma reesei cell derived from T. reesei strain “Rut-C30”, whichis available from the American Type Culture Collection as Trichodermareesei ATCC Deposit No. 56765. In some embodiments, the filamentousfungus is a Trichoderma reesei cell derived from T. reesei strain“RL-P37”, which is available from the culture collection of the NorthernRegional Research Laboratory, US Department of Agriculture as NRRL No.15709.

As used herein, the phrases “variant filamentous fungal cell(s)”,“variant fungal cell(s)”, “variant cell(s)” and the like refer tofilamentous fungal cells that are derived (i.e., obtained) from aparental (control) filamentous fungal cell belonging to thePezizomycotina subphylum. Thus, a “variant” filamentous fungal cell asdefined herein is derived from a “parental” filamentous fungal cell,wherein the “variant” cell comprises at least one genetic modificationwhich is not found in the “parental” cell. For example, when comparing a“variant filamentous fungal cell” vis-à-vis a “parental filamentousfungal cell” of the instant disclosure, the “parental” cell serves asthe genetically unmodified (parental) “control” cell relative to“variant” cell which comprises the at least one genetic modification.

As used herein, the term “genetic modification” refers to thealteration/change of a nucleic acid sequence. The modification caninclude, but is not limited to, a substitution, a deletion, an insertionor a chemical modification of at least one nucleotide in the nucleicacid sequence.

As defined herein, the phrases “variant cell(s) comprising a geneticmodification” and “variant cell(s) comprising a genetic modificationwhich increases the expression of a gene encoding an Ace3-L protein, anAce3-EL protein and/or an Ace3-LN protein”, includes, but is not limitedto, the introduction of at least one copy of a gene or ORF encoding anAce3-L protein, an Ace3-EL protein and/or an Ace3-LN protein into thefilamentous fungal cell. Thus, a filamentous fungal cell comprising theexogenously introduced at least one copy of a gene or ORF encoding theAce3-L protein, the Ace3-EL protein and/or the Ace3-LN protein is avariant fungal cell comprising a genetic modification, relative to theparental fungal cell (which is unmodified).

In other embodiments, parental fungal cells of the disclosure arescreened for the presence of an endogenous ace3 gene encoding any of theAce3 protein disclosed herein (i.e., an ace3 gene encoding an Ace3-Sprotein, an Ace3-SC protein, an Ace3-L protein, an Ace3-LC protein, anAce3-EL protein and an Ace3-LN protein). For example, if a parentalfungal cell is determined to comprise an endogenous copy of an ace3 geneencoding an Ace3-L protein, an Ace3-EL protein or an Ace3-LN protein, avariant fungal cell thereof may be generated by genetic modification,such as, e.g., by replacing the endogenous promoter of ace3 gene with aheterologous promoter. Likewise, if a parental fungal cell is determinedto comprise an endogenous copy of an ace3 gene encoding an Ace3-Sprotein, an Ace3-SC protein or an Ace3-LC protein, a variant fungal cellthereof may be generated by genetic modification, e.g., by introducinginto the fungal cell a polynucleotide construct encoding an Ace3-Lprotein, an Ace3-EL protein and/or an Ace3-LN protein of the disclosure,which may further include genetic modification of the endogenous ace3gene encoding the Ace3-S, Ace3-SC or Ace3-LC protein thereof.

In other embodiments, variant filamentous fungal cells of the disclosurewill comprise further genetic modifications. For example, in certainembodiments, such variant filamentous fungal cells (i.e., comprising anexogenously introduced copy of a gene or ORF encoding an Ace3-L protein,Ace3-EL and/or Ace3-LN protein of the disclosure) further comprise agenetic modification which reduces the expression and/or activity of agene encoding the carbon catabolite repressor protein “Cre1” or the Ace1repressor protein.

In other embodiments, such variant filamentous fungal cells (i.e.,comprising an exogenously introduced copy of a gene or ORF encoding anAce3-L protein, Ace3-EL and/or Ace3-LN protein of the disclosure)further comprise a genetic modification which introduces at least onecopy of a xylanase regulator 1 (Xyr1) set forth in SEQ ID NO: 25 or SEQID NO: 27.

As used herein, an “Ace3-L” protein form (SEQ ID NO: 6) and an “Ace3-LN”protein form (SEQ ID NO: 14; see, FIG. 1B) comprise identical amino acidsequences. However, the headings “Ace3-L” and “Ace3-LN” are used incertain embodiments of the disclosure for comparison with certain genesthereof encoding such protein forms, as is in no way meant to limit thepresent disclosure.

As used herein, the term “host cell” refers to a filamentous fungal cellthat has the capacity to act as a host and expression vehicle for anincoming sequence (i.e., a polynucleotide sequence introduced into thecell), as described herein.

A “heterologous” nucleic acid construct or sequence has a portion of thesequence which is not native or existing in a native form to the cell inwhich it is expressed. Heterologous, with respect to a control sequencerefers to a control sequence (i.e., promoter or enhancer) that does notfunction in nature to regulate the same gene the expression of which itis currently regulating. Generally, heterologous nucleic acid sequencesare not endogenous to the cell or part of the genome in which they arepresent, and have been added to the cell, by infection, transfection,transformation, microinjection, electroporation, or the like. A“heterologous” nucleic acid construct may contain a control sequence/DNAcoding sequence combination that is the same as, or different from acontrol sequence/DNA coding sequence combination found in the nativecell. Similarly, a heterologous protein will often refer to two or moresubsequences that are not found in the same relationship to each otherin nature (e.g., a fusion protein).

As used herein, the term “DNA construct” or “expression construct”refers to a nucleic acid sequence, which comprises at least two DNApolynucleotide fragments. A DNA or expression construct can be used tointroduce nucleic acid sequences into a fungal host cell. The DNA may begenerated in vitro (e.g., by PCR) or any other suitable techniques. Insome preferred embodiments, the DNA construct comprises a sequence ofinterest (e.g., encoding a Ace3-L protein). In certain embodiments, apolynucleotide sequence of interest is operably linked to a promoter. Insome embodiments, the DNA construct further comprises at least oneselectable marker. In further embodiments, the DNA construct comprisessequences homologous to the host cell chromosome. In other embodiments,the DNA construct comprises non-homologous sequences to the host cellchromosome.

As used herein, the terms “cellulase”, “cellulolytic enzymes” or“cellulase enzymes” means bacterial or fungal enzymes such asexoglucanases, exocellobiohydrolases, endoglucanases and/orβ-glucosidases. These different types of cellulase enzymes actsynergistically to convert cellulose and its derivatives to glucose. Forexample, many microbes make enzymes that hydrolyze cellulose, includingthe wood rotting fungus Trichoderma, the compost bacteriaThermomonospora (now Thermobifida), Bacillus, and Cellulomonas;Streptomyces; and the fungi Humicola, Aspergillus and Fusarium. Theenzymes made by these microbes are mixtures of proteins with three typesof actions useful in the conversion of cellulose to glucose:endoglucanases (EG), cellobiohydrolases (CBH), and β-glucosidase (BG).As defined herein, the terms “endoglucanases” (EG), “cellobiohydrolases”(CBH) and “β-glucosidase” (BG) are used interchangeably with theirabbreviations “EG”, “CBH” and “BG”, respectively.

As used herein, the term “carbon limitation” is a state wherein amicroorganism has just enough carbon to produce a desired proteinproduct, but not enough carbon to completely satisfy the organism'srequirement, e.g., sustain growth. Therefore, the maximal amount ofcarbon goes toward protein production.

As used herein, the term “promoter” refers to a nucleic acid sequencethat functions to direct transcription of a downstream gene or an openreading frame (ORF) thereof. The promoter will generally be appropriateto the host cell in which the target gene is being expressed. Thepromoter together with other transcriptional and translationalregulatory nucleic acid sequences (also termed “control sequences”) isnecessary to express a given gene. In general, the transcriptional andtranslational regulatory sequences include, but are not limited to,promoter sequences, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, and enhancer oractivator sequences. In certain embodiments, the promoter is aninducible promoter. In other embodiments, the promoter is a constitutivepromoter.

As used herein, a “promotor sequence” is a DNA sequence which isrecognized by the particular filamentous fungus for expression purposes.A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A “constitutive” promoter is a promoter that is activeunder most environmental and developmental conditions. An “inducible”promoter is a promoter that is active under environmental ordevelopmental regulation. The term “operably linked” refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter, or array of transcription factor binding sites) anda second nucleic acid sequence, wherein the expression control sequencedirects transcription of the nucleic acid corresponding to the secondsequence. Thus, a nucleic acid is “operably linked” when it is placedinto a functional relationship with another nucleic acid sequence. Forexample, DNA encoding a secretory leader (i.e., a signal peptide) isoperably linked to DNA encoding a polypeptide if it is expressed as apre-protein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that the DNAsequences being linked are contiguous, and, in the case of a secretoryleader, contiguous and in reading phase. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice. In other embodiments, linking is accomplished byseamless cloning methods where DNA were joined in a sequence-independentand scar-less manner. The seamless cloning is typically performed with,but not limited to, commercially available systems, such as GibsonAssembly (NEB), NEBuilder HiFi DNA Assembly (NEB), Golden Gate Assembly(NEB), and GeneArt Seamless cloning and Assembly system (ThermoFisherScientific).

As used herein, the term “coding sequence” refers to a nucleotidesequence, which directly specifies the amino acid sequence of its(encoded) protein product. The boundaries of the coding sequence aregenerally determined by an open reading frame, which usually begins withan ATG start codon. The coding sequence typically includes DNA, cDNA,and recombinant nucleotide sequences.

As defined herein, an “open reading frame” (hereinafter, an “ORF”) meansa nucleic acid or nucleic acid sequence (whether naturally occurring,non-naturally occurring, or synthetic) comprising an uninterruptedreading frame consisting of (i) an initiation codon, (ii) a series oftwo (2) of more codons representing amino acids, and (iii) a terminationcodon, the ORF being read (or translated) in the 5′ to 3′ direction.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, that may or may not include regionspreceding and following the coding region (e.g., 5′ untranslated (5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons). The gene may encode commercially important industrial proteinsor peptides, such as enzymes (e.g., proteases, mannanases, xylanases,amylases, glucoamylases, cellulases, oxidases, lipases and the like).The gene of interest may be a naturally occurring gene, a mutated geneor a synthetic gene.

As used herein, the term “recombinant” when used with reference to acell, a nucleic acid, a protein, or a vector, indicates that the cell,nucleic acid, protein or vector, has been modified by the introductionof a heterologous nucleic acid or protein, or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell, orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “vector” is defined herein as a polynucleotide designed tocarry nucleic acid sequences to be introduced into one or more celltypes. Vectors include cloning vectors, expression vectors, shuttlevectors, plasmids, phage or virus particles, DNA constructs, cassettesand the like. Expression vectors may include regulatory sequences suchas promoters, signal sequences, a coding sequences and transcriptionterminators.

An “expression vector” as used herein means a DNA construct comprising acoding sequence that is operably linked to suitable control sequencescapable of effecting expression of a protein in a suitable host. Suchcontrol sequences may include a promoter to effect transcription, anoptional operator sequence to control transcription, a sequence encodingsuitable ribosome binding sites on the mRNA, enhancers and sequenceswhich control termination of transcription and translation.

As used herein, the term “secretory signal sequence” denotes a DNAsequence that encodes a polypeptide (i.e., a “secretory peptide”) that,as a component of a larger polypeptide, directs the larger polypeptidethrough a secretory pathway of a cell in which it is synthesized. Thelarger polypeptide is commonly cleaved to remove the secretory peptideduring transit through the secretory pathway.

As used herein, the term “induction” refers to the increasedtranscription of a gene resulting in the synthesis of a protein ofinterest (hereinafter, a “POI”) in a filamentous fungal cell at amarkedly increased rate in response to the presence of an “inducer”(i.e., inducing substrate). To measure the “induction” of a “gene ofinterest” (hereinafter, a “GOI”) or an “ORF of interest” encoding a POI,variant filamentous fungal (host) cells are treated with a candidateinducing substrate (inducer) and are compared vis-à-vis to parentalfilamentous fungal (control, unmodified) cells which are not treatedwith the inducing substrate (inducer). Thus, the (untreated) parental(control) cells are assigned a relative protein activity value of 100%,wherein induction of the GOI encoding the POI in the variant host cellsis achieved when the activity value (i.e., relative to the controlcells) is greater than 100%, greater than 110%, more preferably 150%,more preferably 200-500% (i.e., two to five fold higher relative to thecontrol), or more preferably 1000-3000% higher.

As used herein, the terms “inducer”, “inducers”, “inducing substrate” or“inducing substrates” are used interchangeably and refer to anycompounds that cause filamentous fungal cells to produce “increasedamounts” of polypeptides (e.g., enzymes, receptors, antibodies and thelike) or other compounds/substances than they would produce if theinducing substrate was absent. Examples of inducing substrates include,but are not limited to, sophorose, lactose, gentibiose and cellulose.

As used herein, the term “inducing feed” refers to a compositioncomprising at least an “inducing substrate” which is fed to filamentousfungal cells, wherein such inducing feed induces the production of aPOI.

As used herein, the term “isolated” or “purified” refers to a nucleicacid or amino acid that is removed from at least one component withwhich it is naturally associated.

As defined herein, the terms “protein of interest” or “POI” refer to apolypeptide that is desired to be expressed in a filamentous fungalcell. Such a protein can be an enzyme, a substrate-binding protein, asurface-active protein, a structural protein, and the like, and can beexpressed at high levels, and can be for the purpose ofcommercialization. The protein of interest can be encoded by anendogenous gene or a heterologous gene (i.e., relative to the variantand/or the parental cells). The protein of interest can be expressedintracellularly or as a secreted (extracellular) protein.

As used herein, the terms “polypeptide” and “protein” (and/or theirrespective plural forms) are used interchangeably to refer to polymersof any length comprising amino acid residues linked by peptide bonds.The conventional one-letter or three-letter codes for amino acidresidues are used herein. The polymer can be linear or branched, it cancomprise modified amino acids, and it can be interrupted by non-aminoacids. The terms also encompass an amino acid polymer that has beenmodified naturally or by intervention (e.g., disulfide bond formation,glycosylation, lipidation, acetylation, phosphorylation, or any othermanipulation or modification, such as conjugation with a labelingcomponent). Also included within the definition are, for example,polypeptides containing one or more analogs of an amino acid (including,for example, unnatural amino acids, etc.), as well as othermodifications known in the art.

As used herein, functionally and/or structurally similar proteins areconsidered to be “related proteins.” Such proteins can be derived fromorganisms of different genera and/or species, or even different classesof organisms (e.g., bacteria and fungi). Related proteins also encompasshomologs determined by primary sequence analysis, determined bysecondary or tertiary structure analysis, or determined by immunologicalcross-reactivity.

As used herein, the phrase “substantially free of an activity,” orsimilar phrases, means that a specified activity is either undetectablein an admixture or present in an amount that would not interfere withthe intended purpose of the admixture.

As used herein, the term “derivative polypeptide” refers to a proteinwhich is derived or derivable from a protein by addition of one or moreamino acids to either or both the N- and C-terminal end(s), substitutionof one or more amino acids at one or a number of different sites in theamino acid sequence, deletion of one or more amino acids at either orboth ends of the protein or at one or more sites in the amino acidsequence, and/or insertion of one or more amino acids at one or moresites in the amino acid sequence. The preparation of a proteinderivative can be achieved by modifying a DNA sequence which encodes forthe native protein, transformation of that DNA sequence into a suitablehost, and expression of the modified DNA sequence to form the derivativeprotein.

Related (and derivative) proteins include “variant proteins.” Variantproteins differ from a reference/parental protein (e.g., a wild-typeprotein) by substitutions, deletions, and/or insertions at a smallnumber of amino acid residues. The number of differing amino acidresidues between the variant and parental protein can be one or more,for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or moreamino acid residues. Variant proteins can share at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or even at least about 99%, or more, amino acidsequence identity with a reference protein. A variant protein can alsodiffer from a reference protein in selected motifs, domains, epitopes,conserved regions, and the like.

As used herein, the term “homologous protein” refers to a protein thathas similar activity and/or structure to a reference protein. It is notintended that homologs necessarily be evolutionarily related. Thus, itis intended that the term encompass the same, similar, or correspondingenzyme(s) (i.e., in terms of structure and function) obtained fromdifferent organisms. In some embodiments, it is desirable to identify ahomolog that has a quaternary, tertiary and/or primary structure similarto the reference protein. In some embodiments, homologous proteinsinduce similar immunological response(s) as a reference protein. In someembodiments, homologous proteins are engineered to produce enzymes withdesired activity(ies).

The degree of homology between sequences can be determined using anysuitable method known in the art (see, e.g., Smith and Waterman, 1981;Needleman and Wunsch, 1970; Pearson and Lipman, 1988; programs such asGAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage (Genetics Computer Group, Madison, Wis.); and Devereux et al.,1984).

As used herein, the phrases “substantially similar” and “substantiallyidentical”, in the context of at least two nucleic acids orpolypeptides, typically means that a polynucleotide or polypeptidecomprises a sequence that has at least about 70% identity, at leastabout 75% identity, at least about 80% identity, at least about 85%identity, at least about 90% identity, at least about 91% identity, atleast about 92% identity, at least about 93% identity, at least about94% identity, at least about 95% identity, at least about 96% identity,at least about 97% identity, at least about 98% identity, or even atleast about 99% identity, or more, compared to the reference (i.e.,wild-type) sequence. Sequence identity can be determined using knownprograms such as BLAST, ALIGN, and CLUSTAL using standard parameters.

As used herein, the term “gene” is synonymous with the term “allele” inreferring to a nucleic acid that encodes and directs the expression of aprotein or RNA. Vegetative forms of filamentous fungi are generallyhaploid, therefore a single copy of a specified gene (i.e., a singleallele) is sufficient to confer a specified phenotype.

As used herein, the terms “wild-type” and “native” are usedinterchangeably and refer to genes, proteins, fungal cells or strains asfound in nature.

As used herein, “deletion of a gene,” refers to its removal from thegenome of a host cell. Where a gene includes control elements (e.g.,enhancer elements) that are not located immediately adjacent to thecoding sequence of a gene, deletion of a gene refers to the deletion ofthe coding sequence, and optionally adjacent enhancer elements,including but not limited to, for example, promoter and/or terminatorsequences.

As used herein, “disruption of a gene” refers broadly to any genetic orchemical manipulation, i.e., mutation, that substantially prevents acell from producing a functional gene product, e.g., a protein, in ahost cell. Exemplary methods of disruption include complete or partialdeletion of any portion of a gene, including a polypeptide-codingsequence, a promoter, an enhancer, or another regulatory element, ormutagenesis of the same, where mutagenesis encompasses substitutions,insertions, deletions, inversions, and combinations and variations,thereof, any of which mutations substantially prevent the production ofa function gene product. A gene can also be disrupted using RNAi,antisense, CRISPR/Cas9 or any other method that abolishes, reduces ormitigates gene expression.

As used herein, the phrase a “variant [host] cell comprising a ‘geneticmodification’ which increases the expression of a gene encoding anAce3-L protein, Ace3-EL and/or Ace3-LN protein of the disclosure”comprises introducing (e.g., via transformation) into the host cell aplasmid or a chromosomal integration cassette comprising an ace3 geneform (or an ORF thereof) encoding such Ace3-L protein, Ace3-EL and/orAce3-LN proteins. In certain other embodiments, such as when a parentalfungal cell natively comprises an endogenous gene form encoding anAce3-L protein, Ace3-EL and/or Ace3-LN protein, the phrase a “variant[host] cell comprising a ‘genetic modification’ which increases theexpression of a gene encoding an Ace3-L protein, Ace3-EL and/or Ace3-LNprotein” includes replacing the native/wild-type ace3 gene promoter witha heterologous promoter.

As used herein, “aerobic fermentation” refers to growth in the presenceof oxygen.

As used herein, the term “cell broth” refers collectively to medium andcells in a liquid/submerged culture.

As used herein, the term “cell mass” refers to the cell component(including intact and lysed cells) present in a liquid/submergedculture. Cell mass can be expressed in dry or wet weight.

As used herein, a “functional polypeptide/protein” is a protein thatpossesses an activity, such as an enzymatic activity, a bindingactivity, a surface-active property, or the like, and which has not beenmutagenized, truncated, or otherwise modified to abolish or reduce thatactivity. Functional polypeptides can be thermostable or thermolabile,as specified.

As used herein, “a functional gene” is a gene capable of being used bycellular components to produce an active gene product, typically aprotein. Functional genes are the antithesis of disrupted genes, whichare modified such that they cannot be used by cellular components toproduce an active gene product, or have a reduced ability to be used bycellular components to produce an active gene product.

As used herein, a “protein of interest” is a protein that is desired tobe produced in a submerged culture of filamentous fungus cells.Generally, proteins of interest are commercially important forindustrial, pharmaceutical, animal health, and food and beverage use,making them desirable to produce in large quantities. Proteins ofinterest are to be distinguished from the myriad other proteinsexpressed by the filamentous fungus cells, which are generally not ofinterest as products and are mainly considered background proteincontaminants.

As used herein, a “variant fungal host cell” produces “substantiallymore protein per unit amount of biomass” than a “parental fungal cell”if the amount of protein produced by the variant cell is at least 5%increased, at least 10% increased, at least 15% increased, or more,compared to the parental cells, wherein the amount of protein isnormalized to the total amount of biomass of cells from which proteinproduction is measured, wherein biomass can be expressed in terms ofeither wet (e.g., of cell pellet) or dry weight.

III. Activator of Cellulase Expression 3 (ace3)

Recently, transcription profiling data from Trichoderma reesei cultures(Hakkinen et al., 2014) in which cellulase/hemicellulase production was“induced” (i.e., via the addition different inducing compositions; e.g.,sophorose, lactose) was examined to identify putative “regulators” ofcellulase and hemicellulase gene expression and a candidate geneencoding a regulatory protein was identified. Hakkinen et al. (2014)identified this candidate gene (see, Hakkinen et al. FIG. 2 and Table 2)as Gene ID No. 77513 (wherein Gene ID Numbers are as in the T. reeseidatabase 2.0) and named the candiate gene “Activator of CellulaseExpression 3” (hereinafter, “ace3”) and the encoded protein (i.e., acandidate transcription factor) “Ace3”. More particularly, the Hakkinenet al. (2014) study used the predicted ace3 ORF (SEQ ID NO: 2), based onthe publicly available genome sequence of T. reesei strain QM6a (see,genome.jgi.doe.gov/Trire2/Trire2.home.html), wherein the QM6a predictedannotation (Gene ID 77513) consists of two exons and one intron (e.g.,see, FIG. 1).

As described herein and further set forth below in the Examples section,Applicants of the instant disclosure discovered surprising andunexpected results when comparing and evaluating the cloned ace3 ORFdescribed in Hakkinen et al., 2014 (i.e., based on the T. reesei “QM6astrain” annotation of Ace3) relative to an ace3 ORF based on the T.reesei “RUT-C30 strain” annotation. For example, as set forth in Example1 below, the T. reesei “QM6a strain” ace3 gene of SEQ ID NO: 1 (and theORF of SEQ ID NO: 2) encodes a shorter Ace3 protein of SEQ ID NO: 3(referred to herein as “Ace3-S”) relative to the T. reesei “RUT-C30strain” ace3 gene of SEQ ID NO: 4 (or ORF of SEQ ID NO: 5), whichencodes a longer Ace3 protein of SEQ ID NO: 6 (referred to herein as“Ace3-L”).

In contrast, the ace3 ORF predicted from the publicly available genomesequence of T. reesei strain Rut-C30 (see,genome.jgi.doe.gov/TrireRUTC30_1/TrireRUTC30_1. home.html) (Gene ID98455) comprises a longer protein sequence (i.e., relative to the Ace3-Sfrom T. reesei QM6a) comprising three exons and two introns (FIG. 1A).More particularly, the start codon predicted by the “RUT-C30” model islocated upstream of that in the “QM6a” model, and there is a non-sensemutation at the C-terminus (Poggi-Parodi et al., 2014), resulting in alonger N-terminal sequence and shorter C-terminal sequence (e.g., seeFIG. 1B).

Likewise, as described in Example 6 below, the position of the 5′ end ofthe ace3 gene coding region is not obvious, and as such, Applicantfurther evaluated the 5′ end of the ace3 gene as described herein. Asbriefly stated above, annotation of the DNA sequence at the Joint GenomeInstitute (JGI) differed between mutant strain Rut-C30 and the wild-typestrain QM6a, even though the DNA sequence is the same. In the QM6a case,the 5′ end of the coding region was suggested to be upstream of exon 3and within intron 2 (as shown in FIG. 11). In the Rut-C30 case, the 5′end of the coding region is within exon 2 (FIG. 11).

Further analysis of the genomic DNA sequence and additional cDNAsequence suggested the possible existence of “exon 1” and “intron 1” (asshown in FIG. 11). In addition, the 3′ end of the ace3 coding region inRut-C30 comprised a mutation creating a premature stop codon, relativeto the sequence of the wild-type isolate QM6a (FIG. 11). Thus, asdescribed in Example 6, Applicant examined the effects ofover-expression of these different possible forms of the ace3 gene asshown in FIG. 12.

Furthermore, as set forth in the Examples below, Applicant constructed(Example 1) and tested (Examples 2-4) the genes encoding the Ace3-Sprotein (SEQ ID NO: 3) and Ace3-L protein (SEQ ID NO: 6) by transformingT. reesei cells with one of four different ace3 expression vectors named“pYL1”, “pYL2”, “pYL3” and “pYL4” (see, FIG. 2A-2D plasmid maps). Morespecifically (Example 1), these expression vectors contain a vectorbackbone with the bacterial ColE1 ori and AmpR gene for replication andselection in E. coli. In addition, the expression vectors (see, FIG.2A-2D) comprise a T. reesei pyr2 selection marker and a heterologous T.reesei promoter sequence (i.e., promoters of hxk1 or pki1) operablylinked to the ace3 ORF coding sequence (ace3-L or ace3-S) with itsnative terminator.

Subsequently, the stable T. reesei transformants generated (i.e.,variant host cells A4-7, B2-1, C2-28 and D3-1) were tested/screened inslow release microtiter plates (srMTPs) (Example 2), shake flasks(Example 3) and small scale fermentation (Example 4) under both“inducing” and “non-inducing” conditions, and the culture supernatantsharvested and analyzed via polyacrylamide gel electrophoresis (see, FIG.3-FIG. 5). As presented in these Examples, all of the host cells tested(i.e., parental, variant A4-7, variant B2-1, variant C2-28 and variantD3-1) secreted a high quantity of proteins in the presence of aninducing substrate (i.e., sophorose or lactose). In contrast, it wassurprisingly observed that in the absence of an inducing substrate(i.e., sophorose or lactose), wherein “glucose” was the sole carbonsource, only the variant host cells expressing the ace3-L ORF (i.e.,variants A4-7 and C2-28) were capable of producing secreted proteins,while the parental (control) cells and variant host cells expressing theace3-S ORF (i.e., variants B2-1 and D3-1) did not produce any detectablesecreted proteins.

In other embodiments, the disclosure further demonstrates enhancedprotein production under “non-inducing” conditions using thirteen (13)different promoters to drive the expression of ace3-L (Example 5)expression constructs. For example, the thirteen promoters testedincluded (i) a formamidase gene (rev3; Protein ID 103041) promoter (SEQID NO: 15), (ii) a J3-xylosidase gene (bxl; Protein ID 121127) promoter(SEQ ID NO: 16), (iii) a transketolase gene (tkl1; Protein ID 2211)promoter (SEQ ID NO: 17), (iv) a gene of unknown function (Protein ID104295) promoter (SEQ ID NO: 18), (v) an oxidoreductase gene (did1;Protein ID 5345) promoter (SEQ ID NO: 19), (vi) a xylanase IV gene(xyn4; Protein ID 111849) promoter (SEQ ID NO: 20), (vii) anα-glucuronidase gene (Protein ID 72526) promoter (SEQ ID NO: 21), (viii)an acetyl xylan esterase gene 1 (axel; Protein ID 73632) promoter (SEQID NO: 22), (ix) a hexose kinase gene (hxk1; Protein ID 73665) promoter(SEQ ID NO: 23), (x) a mitochondrial carrier protein gene (dic1; ProteinID 47930) promoter (SEQ ID NO: 24), (xi) an oligopeptide transportergene (opt; Protein ID 44278) promoter (SEQ ID NO: 25), (xii) a glycerolkinase gene (gut1; Protein ID 58356) promoter (SEQ ID NO: 26) and (xiii)a pyruvate kinase gene (pki1; Protein ID 78439) promoter (SEQ ID NO:27). As shown in Table 3, the parental T. reesei cells only producedsecreted proteins in the presence of the sophorose inducer. In contrast,the variant (daughter) T. reesei cells, comprising and expressing Ace3-Ldriven from any one of thirteen different promoters, produced similaramounts of secreted protein, under both inducing and non-inducingconditions. Also describe in Example 5, the T. reesei parental strainand transformants thereof were further tested in shake flasksexperiments and small scale fermentation. As show in FIG. 8, theparental (control) T. reesei cells only produced secreted proteins inthe presence of the sophorose (“Sop”) inducer, whereas daughter strainLT83 produced similar amounts of secreted protein, under both inducing(“Sop”) and non-inducing (“Glu”) conditions. Likewise, as shown in FIG.9, the parental (control) T. reesei strain only produced secretedproteins in the presence of the sophorose inducer (“Sop”), whereasdaughter strain LT83 produced similar amounts of protein, under bothinducing (“Sop”) and non-inducing (“Glu”) conditions.

Example 6 of the disclosure describes an experimental study of theeffects of over-expressing the different possible forms of the ace3 gene(e.g., see, mutant strain Rut-C30/wild-type strain QM6a genome sequenceannotations discussion above, FIG. 11 and FIG. 12). Thus, the differentforms of ace3 depicted in FIG. 12 were over-expressed in T. reesei,wherein the over-expression vectors for the T. reesei ace3 genes weredesigned to enable targeted integration of ace3 at the glucoamylaselocus (gla1) in T. reesei. Thus, the constructs presented in Table 5differ by having different forms of the ace3 gene. Likewise, the strainsin Table 7 were grown in 24-well microtiter plates in liquid medium witheither 2% lactose or 2% glucose as carbon source, wherein the amount oftotal secreted proteins was measured from the culture supernatants. Inboth media (i.e., 2% lactose or 2% glucose) the over-expression of theace3-L, ace3-EL and ace3-LN forms (i.e., with the RutC-30 C-terminalmutation), improved the production of total proteins (Table 8). In themedium with lactose as the carbon source, over-expression of all theforms of ace3 gene improved the production of total proteins to someextent, but the level of improvement was highest in the strainsover-expressing the ace3-L, ace3-EL and ace3-LN forms of the ace3 gene(Table 8). Thus, it is clear that high levels of secreted protein areobserved under the “non-inducing condition” (i.e., when glucose was usedas a carbon source) when over-expressing the ace3-L, ace3-EL and ace3-LNforms of the ace3 gene.

Example 7 of the disclosure describes a promoter replacement construct(see, FIG. 6) made by fusing a DNA fragment comprising a 5′ regionupstream of the native promoter at the ace3 locus, a loxP-flankedhygromycin B-resistance selectable marker cassette, and a fragmentcomprising a promoter of interest operably fused to the 5′ end of theace3 open reading frame. For example, in certain embodiments, a promoterreplacement construct is used to replace the endogenous ace3 genepromoter in a Trichoderma reesei cell with an alternate promoter.

Example 8 of the disclosure describes replacing an endogenousnon-lignocellulosic gene of interest promoter, with a lignocellulosicgene of interest promoter. For example, a T. reesei glucoamylaseexpression construct was assembled from DNA polynucleotide fragments,wherein an ORF sequence encoding a T. reesei glucoamylase was operablylinked to a 5′ (upstream) T. reesei cbh1 promoter and operably linked toa 3′ (downstream) T. reesei cbh1 terminator, which construct furthercomprised a T. reesei pyr2 gene as selectable marker. The variant(daughter) T. reesei cell (i.e., comprising a genetic modification whichincreases expression of a gene encoding an Ace3-L protein) wastransformed with the glucoamylase expression construct, andtransformants were selected and cultured in liquid medium with glucoseas carbon source (i.e., without an inducing substrate such as sophoroseor lactose) in order to identify those transformants that were able tosecrete the T. reesei glucoamylase enzyme during culture. As presentedin FIG. 10, the parental T. reesei cells produced 1,029 μg/mL ofglucoamylase in defined medium with glucose/sophorose (inducingcondition), and only 38 μg/mL of glucoamylase in defined medium withglucose (non-inducing condition), whereas the modified (daughter) strain“LT88”, comprising ace3-L driven from the dic1 promoter, produced 3-foldhigher glucoamylase under “inducing” (“Sop”) conditions (i.e., relativeto the parental (control) strain), and produced 2.5-fold higherglucoamylase under “non-inducing” (“Glu”) conditions (i.e., relative tothe parental (control) strain). Thus, these results demonstrate that themodified (daughter) cells comprising the Ace3-L ORF not only produceextracellular proteins in the absence of an inducer, but these variantcells also produce more total protein than the parental (control) T.reesei cells under such inducing conditions.

Example 9 describes replacing a natively associated heterologous gene ofinterest promoter with a lignocellulosic gene of interest promoter. Forexample, an ORF encoding Buttiauxella sp. phytase (i.e., a heterologousGOI) is operably linked at the 5′ end to a T. reesei cbh1 promoter andat the 3′ end to a T. reesei cbh1 terminator, wherein the DNA constructfurther comprises a selectable marker. Variant T. reesei cells (i.e.,comprising a genetic modification which increases expression of a geneencoding an Ace3-L protein) is transformed with the phytase expressionconstruct, transformants are selected and cultured in liquid medium withglucose as carbon source (i.e., without an inducing substrate such assophorose or lactose) in order to identify those transformants that areable to secrete Buttiauxella phytase enzyme during culture.

Example 10 of the disclosure describes the construction of native ace3promoter replacement vectors, which vectors contained a Streptococcuspyogenes cas9 gene, expressed under the T. reesei pki1 promoter andguide RNA expressed under a U6 promoter. For example, the cas9 mediatedace3 promoter replacement vectors (pCHL760 and pCHL761) were transformedinto T. reesei parental cells, and to test the functionality of ace3promoter replaced strains, cells were grown in the presence and absenceof an inducer substrate (sophorose) in 50 ml submerged culture in shakeflasks. As seen on SDS-PAGE, parental cells (FIG. 23, ID 1275.8.1)produced much less secreted protein in defined medium with glucose(non-inducing) compared to glucose/sophorose (induction). In contrast,transformants 2218, 2219, 2220, 2222 and 2223 produced similar amountsof secreted protein under inducing and non-inducing conditions,demonstrating that the variant cells harboring the hxk1 or dic1 promoter(i.e., replacing the native ace3 promoter at ace3 locus) producedextracellular proteins in the absence of an inducer.

Thus, as contemplated and described herein, certain aspects of thepresent disclosure are directed to the production of one or moreendogenous filamentous fungal lignocellulosic degrading enzymes (i.e.,cellulolytic enzymes, e.g., a cellobiohydrolase, a xylanase, anendoglucanase and the like). More specifically, certain embodiments ofthe disclosure are directed to producing such endogenous enzymes in avariant host cell of the disclosure (i.e., a variant host cellcomprising a genetic modification which increases expression of anAce3-L protein, Ace3-EL and/or Ace3-LN protein), in the complete absenceof an inducing substrate. The variant host cells, compositions andmethods of the instant disclosure are of particular utility forsignificantly reducing the cost/expense of producing the aforementionedcellulolytic enzymes, particularly due to the fact such variant hostcells of the disclosure do not require an inducing substrate to producesuch cellulolytic enzymes (i.e., in contrast to the parental cells whichonly produce such cellulolytic enzymes in presence of inducingsubstrates).

For example, in certain embodiments, the disclosure is directed tovariant fungal host cells capable of expressing/producing one or moreendogenous proteins of interest in the absence of an inducing substrateand/or one or more heterologous proteins of interest in the absence ofan inducing substrate. Therefore, in certain embodiments, a variantfungal host cell of the disclosure (i.e., comprising a geneticmodification which increases the expression of Ace3-L protein, Ace3-ELand/or Ace3-LN) is further modified to express an endogenous,non-lignocellulosic protein of interest and/or a heterologous protein ofinterest. For example, in certain embodiments, a gene encoding anendogenous, non-lignocellulosic protein of interest is modified in thevariant fungal host cell. Thus, in certain embodiments, the promoternatively associated with a gene (or ORF) encoding an endogenous,non-lignocellulosic protein of interest is replaced with a promoter froma filamentous fungi gene encoding a lignocellulosic protein (e.g., a5′-lignocellulosic gene promoter operably linked to an endogenous geneencoding a non-lignocellulosic protein of interest). Likewise, incertain other embodiments, a variant fungal host cell of the disclosure(i.e., comprising a genetic modification which increases the expressionof Ace3-L protein, Ace3-EL and/or Ace3-LN) is modified to express aheterologous protein of interest. Thus, in certain other embodiments,the promoter natively associated with a gene encoding a heterologousprotein of interest is replaced with a promoter from a filamentous fungigene encoding a lignocellulosic protein (e.g., a 5′-lignocellulosic genepromoter operably linked to a heterologous gene encoding a heterologousprotein of interest).

Thus, in certain embodiments, the instant disclosure is directed to avariant filamentous fungal cell derived from a parental filamentousfungal cell, wherein the variant cell comprises a genetic modificationwhich increases the expression of a gene encoding an Ace-L proteinrelative to the parental cell, wherein the encoded Ace3-L proteincomprises about 90% sequence identity to the Ace3-L protein of SEQ IDNO: 6. For example, in certain embodiments, an encoded Ace3 proteincomprising about 90% sequence identity to SEQ ID NO: 6, comprises“Lys-Ala-Ser-Asp” as the last four C-terminal amino acids. In otherembodiments, an encoded Ace3 protein comprising about 90% sequenceidentity to SEQ ID NO: 6, further comprises an N-terminal amino acidfragment of SEQ ID NO: 98 operably linked and preceding SEQ ID NO: 6. Inanother embodiment, an Ace-3 protein comprises about 90% sequenceidentity to SEQ ID NO: 12.

In certain other embodiments, a polynucleotide of the disclosurecomprises a nucleotide sequence comprising about 90% sequence identityto SEQ ID NO: 4, SEQ ID NO: 11 or SEQ ID NO: 13. In other embodiments, apolynucleotide of the disclosure comprises a nucleotide sequencecomprising about 90% sequence identity to SEQ ID NO: 5, SEQ ID NO: 101or SEQ ID NO: 102.

IV. Filamentous Fungal Host Cells

In certain embodiments of the disclosure, variant filamentous fungalcells (i.e., derived from parental filamentous fungal cells) areprovided which comprise a genetic modification which increases theexpression of a gene or ORF encoding an Ace3-L polypeptide. Moreparticularly, in certain embodiments, variant filamentous fungal cells(i.e., relative to the parental (control) cells) comprise a geneticmodification that increases the expression of a gene (or ORF) encodingan Ace3-L protein of SEQ ID NO: 6. In preferred embodiments, suchvariant fungal cells comprising a genetic modification which increasesthe expression of a gene (or ORF) encoding an Ace3-L protein of SEQ IDNO: 6 are capable of producing at least one endogenous protein ofinterest in the absence of an inducing substrate. In other embodiments,such variant fungal cells comprising a genetic modification whichincreases the expression of a gene (or ORF) encoding an Ace3-L proteinof SEQ ID NO: 6 are capable of producing at least one heterologousprotein of interest in the absence of an inducing substrate.

Thus, in certain embodiments, a filamentous fungal cell for manipulationand use in the present disclosure includes filamentous fungi from thephylum Ascomycota, subphylum Pezizomycotina, particularly fungi thathave a vegetative hyphae state. Such organisms include filamentousfungal cells used for the production of commercially importantindustrial and pharmaceutical proteins, including, but not limited toTrichoderma spp., Aspergillus spp., Fusarium spp., Scedosporium spp.,Penicillium spp., Chrysosporium spp., Cephalosporium spp., Talaromycesspp., Geosmithia spp., Myceliophthora spp. and Neurospora spp.

Particular filamentous fungi include, but are not limited to,Trichoderma reesei (previously classified as Trichoderma longibrachiatumand Hypocrea jecorina), Aspergillus niger, Aspergillus fumigatus,Aspergillus itaconicus, Aspergillus oryzae, Aspergillus nidulans,Aspergillus terreus, Aspergillus sojae, Aspergillus japonicus,Scedosporium prolificans, Neurospora crassa, Penicillium funiculosum,Penicillium chrysogenum, Talaromyces (Geosmithia) emersonii, Fusariumvenenatum, Myceliophthora thermophila and Chrysosporium lucknowense.

V. Recombinant Nucleic Acids and Molecular Biology

In certain embodiments, the instant disclosure is directed to variantfilamentous fungal host cells comprising a genetic modification whichincreases the expression of a gene or ORF encoding an Ace3-L, Ace3-EL orAce3-LN protein. As set forth above, such variant host cells are capableof producing one or more proteins of interest in the absence of aninducing substrate (i.e., in contrast to the unmodified parental(control) cells).

Thus, in certain embodiments, the disclosure is directed to recombinantnucleic acids comprising a gene or ORF encoding an Ace3-L, Ace3-EL orAce3-LN protein. In certain embodiments, a recombinant nucleic acidcomprises a polynucleotide expression cassette for production of anAce3-L, Ace3-EL or Ace3-LN protein in a filamentous fungal host cell. Inother embodiments, the polynucleotide expression cassette is comprisedwithin an expression vector. In certain embodiments, the expressionvector is a plasmid. In other embodiments, the recombinant nucleic acid,polynucleotide expression cassette or expression vector thereofcomprises a nucleotide sequence comprising at least 85% sequenceidentity to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 101,SEQ ID NO: 13 or SEQ ID NO: 102. In another embodiment, the recombinantnucleic acid, polynucleotide expression cassette or expression vectorthereof comprises a nucleotide sequence encoding an Ace3-L proteincomprising about 90% sequence identity to SEQ ID NO: 6.

In certain other embodiments, the recombinant nucleic acid (orpolynucleotide expression cassette thereof or expression vector thereof)further comprises one or more selectable markers. Selectable markers foruse in filamentous fungi include, but are not limited to, alsl, amdS,hygR, pyr2, pyr4, pyrG, sucA, a bleomycin resistance marker, ablasticidin resistance marker, a pyrithiamine resistance marker, achlorimuron ethyl resistance marker, a neomycin resistance marker, anadenine pathway gene, a tryptophan pathway gene, a thymidine kinasemarker and the like. In a particular embodiment, the selectable markeris pyr2, which compositions and methods of use are generally set forthin PCT Publication No. WO2011/153449. Thus, in certain embodiments, apolynucleotide construct encoding an Ace3 protein of the disclosurecomprises a nucleic acid sequence encoding a selectable marker operablylinked thereto.

In another embodiment, the recombinant nucleic acid, polynucleotideconstruct, polynucleotide expression cassette or expression vectorthereof comprises a heterologous promoter driving the expression of thegene (or ORF) encoding an Ace3-L, Ace3-EL or Ace3-LN protein. Moreparticularly, in certain embodiments, the heterologous promoter is aconstitutive or an inducible promoter. In particular embodiments, aheterologous promoter is selected from the group consisting of a rev3promoter, a bxl promoter, a tkl1 promoter, a PID104295 promoter, a dld1promoter, a xyn4 promoter, a PID72526 promoter, an axe1 promoter, a hxk1promoter, a dic1 promoter, an opt promoter, a gut1 promoter and apki1promoter. Without wishing to be bound by a particular theory ormechanism of action, it is contemplated herein that promoters such asrev3, bxl, tkl, PID104295, dld1, xyn4, PID72526, axe1, hxk1, dic1, opt,gut1 and a pki1, which yield higher expression levels under glucoselimiting conditions (i.e., vis-à-vis excess glucose concentrations),have particular utility in the instant disclosure. Thus, in certainembodiments, a recombinant nucleic acid (or polynucleotide construct,polynucleotide expression cassette or expression vector thereof)comprises a promoter which is 5′ and operably linked to the nucleic acidsequence encoding the Ace3 protein.

In another embodiment, a recombinant nucleic acid (or polynucleotideconstruct, polynucleotide expression cassette or expression vectorthereof) further comprises a nucleic acid sequence encoding a nativeace3 terminator sequence. Thus, in certain embodiments, a recombinantnucleic acid (or polynucleotide construct, polynucleotide expressioncassette, or expression vector thereof) comprises a promoter which is 5′and operably linked to a nucleic acid sequence encoding an Ace3 proteinand a native ace3 terminator sequence which is 3′ and operably linked toa nucleic acid sequence encoding an Ace3 protein (e.g.,5′-Pro-ORF-Term-3′, where “Pro” is a constitutive promoter, “ORF”encodes Ace3 and “Term” is a native ace3 terminator sequence).

Thus, in certain embodiments, standard techniques for transformation offilamentous fungi and culturing the fungi (which are well known to oneskilled in the art) are used to transform a fungal host cell of thedisclosure. Thus, the introduction of a DNA construct or vector into afungal host cell includes techniques such as transformation,electroporation, nuclear microinjection, transduction, transfection(e.g., lipofection mediated and DEAE-Dextrin mediated transfection),incubation with calcium phosphate DNA precipitate, high velocitybombardment with DNA-coated microprojectiles, gene gun or biolistictransformation, protoplast fusion and the like. General transformationtechniques are known in the art (see, e.g., Ausubel et al., 1987,Sambrook et al., 2001 and 2012, and Campbell et al., 1989). Theexpression of heterologous proteins in Trichoderma is described, forexample, in U.S. Pat. Nos. 6,022,725; 6,268,328; Harkki et al., 1991 andHarkki et al., 1989. Reference is also made to Cao et al. (2000), fortransformation of Aspergillus strains.

Generally, transformation of Trichoderma sp. uses protoplasts or cellsthat have been subjected to a permeability treatment, typically at adensity of 10⁵ to 10⁷/mL, particularly 2×10⁶/mL. A volume of 100 μL ofthese protoplasts or cells in an appropriate solution (e.g., 1.2 Msorbitol and 50 mM CaCl₂) is mixed with the desired DNA. Generally, ahigh concentration of polyethylene glycol (PEG) is added to the uptakesolution. Additives, such as dimethyl sulfoxide, heparin, spermidine,potassium chloride and the like, may also be added to the uptakesolution to facilitate transformation. Similar procedures are availablefor other fungal host cells. See, e.g., U.S. Pat. Nos. 6,022,725 and6,268,328, both of which are incorporated by reference.

In certain embodiments, the instant disclosure is directed to theexpression and production of one or more proteins of interest which areendogenous to the filamentous fungal host cell (i.e., the endogenousproteins are produced by a variant fungal host cell of the disclosurecomprising a genetic modification which increasers expression ofAce3-L). In other embodiments, the disclosure is directed to expressingand producing one or more proteins of interest which are heterologous tothe to the filamentous fungal host cell. Therefore, the instantdisclosure generally relies on routine techniques in the field ofrecombinant genetics. Basic texts disclosing the general methods of usein present disclosure include Sambrook et al., (2^(nd) Edition, 1989);Kriegler (1990) and Ausubel et al., (1994).

Thus, in certain embodiments, a heterologous gene or ORF encoding aprotein of interest is introduced into a filamentous fungal (host) cell.In certain embodiments, the heterologous gene or ORF is typically clonedinto an intermediate vector, before being transformed into a filamentousfungal (host) cells for replication and/or expression. Theseintermediate vectors can be prokaryotic vectors, such as, e.g.,plasmids, or shuttle vectors. In certain embodiments, the expression ofthe heterologous gene or ORF is under the control of its nativepromoter. In other embodiments, the expression of the heterologous geneor ORF is placed under the control of a heterologous promoter, which canbe a heterologous constitutive promoter or a heterologous induciblepromoter.

Those skilled in the art are aware that a natural (native) promoter canbe modified by replacement, substitution, addition or elimination of oneor more nucleotides, without changing its function. The practice of theinvention encompasses but is not constrained by such alterations to thepromoter.

The expression vector/construct typically contains a transcription unitor expression cassette that contains all the additional elementsrequired for the expression of the heterologous sequence. For example, atypical expression cassette contains a 5′ promoter operably linked tothe heterologous nucleic acid sequence encoding a protein of interestand may further comprise sequence signals required for efficientpolyadenylation of the transcript, ribosome binding sites, andtranslation termination. Additional elements of the cassette may includeenhancers and, if genomic DNA is used as the structural gene, intronswith functional splice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette may alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes. Although any fungal terminator is likely to befunctional in the present invention, preferred terminators include: theterminator from Trichoderma cbhI gene, the terminator from Aspergillusnidulans trpC gene (Yelton et al., 1984; Mullaney et al., 1985), theAspergillus awamori or Aspergillus niger glucoamylase genes (Nunberg etal., 1984; Boel et al., 1984) and/or the Mucor miehei carboxyl proteasegene (EPO Publication No. 0215594).

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includebacteriophages 2 and M13, as well as plasmids such as pBR322 basedplasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST,and LacZ. Epitope tags can also be added to recombinant proteins toprovide convenient methods of isolation, e.g., c-myc.

The elements that can be included in expression vectors may also be areplicon, a gene encoding antibiotic resistance to permit selection ofbacteria that harbor recombinant plasmids, or unique restriction sitesin nonessential regions of the plasmid to allow insertion ofheterologous sequences. The particular antibiotic resistance gene chosenis not dispositive either, as any of the many resistance genes known inthe art may be suitable. The prokaryotic sequences are preferably chosensuch that they do not interfere with the replication or integration ofthe DNA in Trichoderma reesei.

The methods of transformation of the present invention may result in thestable integration of all or part of the transformation vector into thegenome of the filamentous fungus. However, transformation resulting inthe maintenance of a self-replicating extra-chromosomal transformationvector is also contemplated.

Many standard transfection methods can be used to produce Trichodermareesei cell lines that express large quantities of the heterologusprotein. Some of the published methods for the introduction of DNAconstructs into cellulase-producing strains of Trichoderma includeLorito, Hayes, DiPietro and Harman, 1993, Curr. Genet. 24: 349-356;Goldman, VanMontagu and Herrera-Estrella, 1990, Curr. Genet. 17:169-174;Penttila, Nevalainen, Ratto, Salminen and Knowles, 1987, Gene 6:155-164, for Aspergillus Yelton, Hamer and Timberlake, 1984, Proc. Natl.Acad. Sci. USA 81: 1470-1474, for Fusarium Bajar, Podila andKolattukudy, 1991, Proc. Natl. Acad. Sci. USA 88: 8202-8212, forStreptomyces Hopwood et al., 1985, The John Innes Foundation, Norwich,UK and for Bacillus Brigidi, DeRossi, Bertarini, Riccardi and Matteuzzi,1990, FEMS Microbiol. Lett. 55: 135-138).

Any of the known procedures for introducing foreign nucleotide sequencesinto host cells may be used. These include the use of calcium phosphatetransfection, polybrene, protoplast fusion, electroporation, biolistics,liposomes, microinjection, plasma vectors, viral vectors and any of theother known methods for introducing cloned genomic DNA, cDNA, syntheticDNA or other foreign genetic material into a host cell (see, e.g.,Sambrook et al., supra). Also of use is the Agrobacterium-mediatedtransfection method such as the one described in U.S. Pat. No.6,255,115. It is only necessary that the particular genetic engineeringprocedure used be capable of successfully introducing at least one geneinto the host cell capable of expressing the heterologous gene.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofgenes under control of cellulase gene promoter sequences. Large batchesof transformed cells can be cultured as described herein. Finally,product is recovered from the culture using standard techniques.

Thus, the invention herein provides for the expression and enhancedsecretion of desired polypeptides whose expression is under control ofcellulase gene promoter sequences including naturally occurringcellulase genes, fusion DNA sequences, and various heterologousconstructs. The invention also provides processes for expressing andsecreting high levels of such desired

VI. Proteins of Interest

As stated above, certain embodiments of the disclosure are directed tovariant filamentous fungal cells (derived from a parental filamentousfungal cells), wherein the variant cells comprise a genetic modificationwhich increases the expression of a gene encoding an Ace3-L, Ace3-EL orAce3-LN protein (relative to the parental cell), wherein the encodedAce3-L, Ace3-EL or Ace3-LN protein comprises about 90% sequence identityto the Ace3-L protein of SEQ ID NO: 6 and wherein the variant cellsexpress at least one protein of interest (POI) in the absence of aninducing substrate.

Certain embodiments of the present disclosure are particularly usefulfor enhancing the intracellular and/or extracellular production ofproteins (i.e., proteins of interest) in the absence of an inducingsubstrate. The protein of interest may be an endogenous protein (i.e.,endogenous in the host cell) or a heterologous protein (i.e., not nativein the host cell). Proteins that can be produced according to theinstant disclosure include, but are not limited to, hormones, enzymes,growth factors, cytokines, antibodies and the like.

For example, a protein of interest can include, but is not limited to, ahemicellulase, a peroxidases, a protease, a cellulase, a xylanase, alipase, a phospholipase, an esterase, a cutinase, a pectinase, akeratinase, a reductase, an oxidase, a phenol oxidase, a lipoxygenase, aligninase, a pullulanase, a tannase, a pentosanase, a mannanase, aβ-glucanase, a hyaluronidase, a chondroitinase, a laccase, a amylase, aglucoamylase, an acetyl esterase, an aminopeptidase, amylases, anarabinases, an arabinosidase, an arabinofuranosidase, acarboxypeptidase, a catalase, a deoxyribonuclease, an epimerase, anα-galactosidase, a β-galactosidase, an α-glucanases, a glucan lysase, anendo-β-glucanase, a glucose oxidase, a glucuronidase, an invertase, anisomerase, and the like.

In certain embodiments, a protein of interest includes, but is notlimited to, enzymes disclosed in PCT Application Publication Nos.WO03/027306, WO200352118, WO200352054, WO200352057, WO200352055,WO200352056, WO200416760, WO9210581, WO200448592, WO200443980,WO200528636, WO200501065, WO2005/001036, WO2005/093050, WO200593073,WO200674005, WO2009/149202, WO2011/038019, WO2010/141779, WO2011/063308,WO2012/125951, WO2012/125925, WO2012125937, WO/2011/153276,WO2014/093275, WO2014/070837, WO2014/070841, WO2014/070844,WO2014/093281, WO2014/093282, WO2014/093287, WO2014/093294,WO2015/084596 and WO2016/069541.

Optimal conditions for the production of the proteins will vary with thechoice of the host cell, and with the choice of the protein(s) to beexpressed. Such conditions may be readily ascertained by one skilled inthe art through routine experimentation and/or optimization.

The protein of interest can be purified or isolated after expression.The protein of interest may be isolated or purified in a variety of waysknown to those skilled in the art depending on what other components arepresent in the sample. Standard purification methods includeelectrophoretic, molecular, immunological and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocusing. For example,the protein of interest may be purified using a standard anti-protein ofinterest antibody column Ultrafiltration and diafiltration techniques,in conjunction with protein concentration, are also useful. The degreeof purification necessary will vary depending on the intended use of theprotein of interest. In certain instances, no purification of theprotein will be necessary.

In certain other embodiments, to confirm that a genetically modifiedfungal cell of the disclosure (i.e., a variant fungal host cellcomprising a genetic modification which increases the expression oface3-L) has the capability of producing an increased level of a proteinof interest, various methods of screening may be performed. Theexpression vector may encode a polypeptide fusion to the target proteinwhich serves as a detectable label or the target protein itself mayserve as the selectable or screenable marker. The labeled protein may bedetected via western blotting, dot blotting (methods available at theCold Spring Harbor Protocols website), ELISA, or, if the label is GFP,whole cell fluorescence or FACS. For example, a 6-histidine tag would beincluded as a fusion to the target protein, and this tag would bedetected by western blotting. If the target protein expresses atsufficiently high levels, SDS-PAGE combined with Coomassie/silverstaining, may be performed to detect increases in variant host cellexpression over parental (control) cell, in which case no label isnecessary. In addition, other methods may be used to confirm theimproved level of a protein of interest, such as, the detection of theincrease of protein activity or amount per cell, protein activity oramount per milliliter of medium, allowing cultures or fermentations tocontinue efficiently for longer periods of time, or through acombination of these methods.

The detection of specific productivity is another method to evaluate theprotein production. Specific productivity (Qp) can be determined by thefollowing equation:

Qp=gP/gDCW·hr

wherein “gP” is grams of protein produced in the tank, “gDCW” is gramsof dry cell weight (DCW) in the tank, “hr” is fermentation time in hoursfrom the time of inoculation, which include the time of production aswell as growth time.

In other embodiments, the variant fungal host cell is capable ofproducing at least about 0.5%, for example, at least about 0.5%, atleast about 0.7%, at least about 1%, at least about 1.5%, at least about2.0%, at least about 2.5%, or even at least about 3%, or more of aprotein of interest, as compared vis-à-vis to the (unmodified) parentalcell.

VII. Fermentation

In certain embodiments, the present disclosure provides methods ofproducing a protein of interest comprising fermenting a variant fungalcell, wherein the variant fungal cell secrets the protein of interest.In general, fermentation methods well known in the art are used toferment the variant fungal cells. In some embodiments, the fungal cellsare grown under batch or continuous fermentation conditions. A classicalbatch fermentation is a closed system, where the composition of themedium is set at the beginning of the fermentation and is not alteredduring the fermentation. At the beginning of the fermentation, themedium is inoculated with the desired organism(s). In this method,fermentation is permitted to occur without the addition of anycomponents to the system. Typically, a batch fermentation qualifies as a“batch” with respect to the addition of the carbon source, and attemptsare often made to control factors such as pH and oxygen concentration.The metabolite and biomass compositions of the batch system changeconstantly up to the time the fermentation is stopped. Within batchcultures, cells progress through a static lag phase to a high growth logphase and finally to a stationary phase, where growth rate is diminishedor halted. If untreated, cells in the stationary phase eventually die.In general, cells in log phase are responsible for the bulk ofproduction of product.

A suitable variation on the standard batch system is the “fed-batchfermentation” system. In this variation of a typical batch system, thesubstrate is added in increments as the fermentation progresses.Fed-batch systems are useful when catabolite repression likely inhibitsthe metabolism of the cells and where it is desirable to have limitedamounts of substrate in the medium. Measurement of the actual substrateconcentration in fed-batch systems is difficult and is thereforeestimated on the basis of the changes of measurable factors, such as pH,dissolved oxygen and the partial pressure of waste gases, such as CO₂.Batch and fed-batch fermentations are common and well known in the art.

Continuous fermentation is an open system where a defined fermentationmedium is added continuously to a bioreactor, and an equal amount ofconditioned medium is removed simultaneously for processing. Continuousfermentation generally maintains the cultures at a constant highdensity, where cells are primarily in log phase growth. Continuousfermentation allows for the modulation of one or more factors thataffect cell growth and/or product concentration. For example, in oneembodiment, a limiting nutrient, such as the carbon source or nitrogensource, is maintained at a fixed rate and all other parameters areallowed to moderate. In other systems, a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Continuous systems striveto maintain steady state growth conditions. Thus, cell loss due tomedium being drawn off should be balanced against the cell growth ratein the fermentation. Methods of modulating nutrients and growth factorsfor continuous fermentation processes, as well as techniques formaximizing the rate of product formation, are well known in the art ofindustrial microbiology.

Certain embodiments of the instant disclosure are related tofermentation procedures for culturing fungi. Fermentation procedures forproduction of cellulase enzymes are known in the art. For example,cellulase enzymes can be produced either by solid or submerged culture,including batch, fed-batch and continuous-flow processes. Culturing isgenerally accomplished in a growth medium comprising an aqueous mineralsalts medium, organic growth factors, a carbon and energy sourcematerial, molecular oxygen, and, of course, a starting inoculum of thefilamentous fungal host to be employed.

In addition to the carbon and energy source, oxygen, assimilablenitrogen, and an inoculum of the microorganism, it is necessary tosupply suitable amounts in proper proportions of mineral nutrients toassure proper microorganism growth, maximize the assimilation of thecarbon and energy source by the cells in the microbial conversionprocess, and achieve maximum cellular yields with maximum cell densityin the fermentation media.

The composition of the aqueous mineral medium can vary over a widerange, depending in part on the microorganism and substrate employed, asis known in the art. The mineral media should include, in addition tonitrogen, suitable amounts of phosphorus, magnesium, calcium, potassium,sulfur, and sodium, in suitable soluble assimilable ionic and combinedforms, and also present preferably should be certain trace elements suchas copper, manganese, molybdenum, zinc, iron, boron, and iodine, andothers, again in suitable soluble assimilable form, all as known in theart.

The fermentation reaction is an aerobic process in which the molecularoxygen needed is supplied by a molecular oxygen-containing gas such asair, oxygen-enriched air, or even substantially pure molecular oxygen,provided to maintain the contents of the fermentation vessel with asuitable oxygen partial pressure effective in assisting themicroorganism species to grow in a thriving fashion.

The fermentation temperature can vary somewhat, but for filamentousfungi such as Trichoderma reesei, the temperature generally will bewithin the range of about 20° C. to 40° C., generally preferably in therange of about 25° C. to 34° C.

The microorganisms also require a source of assimilable nitrogen. Thesource of assimilable nitrogen can be any nitrogen-containing compoundor compounds capable of releasing nitrogen in a form suitable formetabolic utilization by the microorganism. While a variety of organicnitrogen source compounds, such as protein hydrolysates, can beemployed, usually cheap nitrogen-containing compounds such as ammonia,ammonium hydroxide, urea, and various ammonium salts such as ammoniumphosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride,or various other ammonium compounds can be utilized Ammonia gas itselfis convenient for large scale operations, and can be employed bybubbling through the aqueous ferment (fermentation medium) in suitableamounts. At the same time, such ammonia can also be employed to assistin pH control.

The pH range in the aqueous microbial ferment (fermentation admixture)should be in the exemplary range of about 2.0 to 8.0. With filamentousfungi, the pH normally is within the range of about 2.5 to 8.0; withTrichoderma reesei, the pH normally is within the range of about 3.0 to7.0. Preferences for pH range of microorganisms are dependent on themedia employed to some extent, as well as the particular microorganism,and thus change somewhat with change in media as can be readilydetermined by those skilled in the art.

Preferably, the fermentation is conducted in such a manner that thecarbon-containing substrate can be controlled as a limiting factor,thereby providing good conversion of the carbon-containing substrate tocells and avoiding contamination of the cells with a substantial amountof unconverted substrate. The latter is not a problem with water-solublesubstrates, since any remaining traces are readily washed off. It may bea problem, however, in the case of non-water-soluble substrates, andrequire added product-treatment steps such as suitable washing steps.

As described above, the time to reach this level is not critical and mayvary with the particular microorganism and fermentation process beingconducted. However, it is well known in the art how to determine thecarbon source concentration in the fermentation medium and whether ornot the desired level of carbon source has been achieved.

The fermentation can be conducted as a batch or continuous operation,fed batch operation is much to be preferred for ease of control,production of uniform quantities of products, and most economical usesof all equipment.

If desired, part or all of the carbon and energy source material and/orpart of the assimilable nitrogen source such as ammonia can be added tothe aqueous mineral medium prior to feeding the aqueous mineral mediumto the fermenter.

Each of the streams introduced into the reactor preferably is controlledat a predetermined rate, or in response to a need determinable bymonitoring such as concentration of the carbon and energy substrate, pH,dissolved oxygen, oxygen or carbon dioxide in the off-gases from thefermenter, cell density measurable by dry cell weights, lighttransmittancy, or the like. The feed rates of the various materials canbe varied so as to obtain as rapid a cell growth rate as possible,consistent with efficient utilization of the carbon and energy source,to obtain as high a yield of microorganism cells relative to substratecharge as possible.

In either a batch, or the preferred fed batch operation, all equipment,reactor, or fermentation means, vessel or container, piping, attendantcirculating or cooling devices, and the like, are initially sterilized,usually by employing steam such as at about 121° C. for at least about15 minutes. The sterilized reactor then is inoculated with a culture ofthe selected microorganism in the presence of all the requirednutrients, including oxygen, and the carbon-containing substrate. Thetype of fermenter employed is not critical.

The collection and purification of (e.g., cellulase) enzymes from thefermentation broth can also be done by procedures known to one of skillin the art. The fermentation broth will generally contain cellulardebris, including cells, various suspended solids and other biomasscontaminants, as well as the desired cellulase enzyme product, which arepreferably removed from the fermentation broth by means known in theart.

Suitable processes for such removal include conventional solid-liquidseparation techniques such as, e.g., centrifugation, filtration,dialysis, microfiltration, rotary vacuum filtration, or other knownprocesses, to produce a cell-free filtrate. It may be preferable tofurther concentrate the fermentation broth or the cell-free filtrateprior to crystallization using techniques such as ultrafiltration,evaporation or precipitation.

Precipitating the proteinaceous components of the supernatant orfiltrate may be accomplished by means of a salt, e.g., ammonium sulfate,followed by purification by a variety of chromatographic procedures,e.g., ion exchange chromatography, affinity chromatography or similarart recognized procedures.

EXAMPLES

It should be understood that the following Examples, while indicatingembodiments of the disclosure, are given by way of illustration only.From the above discussion and these Examples, one of skill in the artcan make various changes and modifications of the disclosure to adapt itto various usages and conditions. Such modifications are also intendedto fall within the scope of the claimed invention.

Example 1 Generation of Ace3 Over Expression in Filamentous Fungal Cells1A. Overview

In the present example, variant Trichoderma reesei cells (i.e., anexemplary filamentous fungi) expressing an ace3 gene were generated bytransforming parental T. reesei cells with a nucleic acid containing thepyr2 gene, a heterologous promoter and an ace3 gene, using protoplasttransformation. As generally presented in FIG. 1 and FIG. 2, four (4)different ace3-expression vectors were constructed (FIG. 2A-2D) with two(2) different promoters and two different versions of ace3 ORF (FIG. 1;ace3-SC and ace3-L) in four different combinations. Promoters of hxk1(gene encoding hexokinase) and pki1 (gene encoding pyruvate kinase) wereselected to drive constitutive expression of ace3, however, otherpromoters can also be used and selected by the skilled artisan.

1B. Trichoderma reesei Host Cells

The T. reesei parental host cells set forth in the following exampleswere derived from T. reesei strain RL-P37 (NRRL Deposit No. 15709),wherein the T. reesei pyr2 gene has been deleted, as generally describedby Sheir-Neiss and Montenecourt, 1984.

1C. Construction of Ace3 Expression Vectors

As set forth in the Detailed Description of the disclosure above, Ace3is a T. reesei transcriptional factor recently shown to be required forcellulase and hemi-cellulase production under inducing conditions (i.e.,in the presence of lactose) (Hakkinen et al., 2014). More particularly,Hakkinen et al. (2014) used the predicted ace3 ORF, based on thepublicly available genome sequence of T. reesei strain QM6a (see,genome.jgi.doe.gov/Trire2/Trire2.home.html), wherein the QM6a predictedannotation (Protein ID 77513) consists of two exons and one intron(e.g., see, FIG. 1).

In addition, the ace3 ORF predicted from the publicly available genomesequence of T. reesei strain Rut-C30 (see,(genome.jgi.doe.gov/TrireRUTC30_1/TrireRUTC30_1. home.html) (Protein ID98455) comprises a longer protein sequence (i.e., relative to the(short) ace3 from T. reesei QM6a) comprising three exons and two introns(FIG. 1). More particularly, the start codon predicted by the “RUT-C30”model is located upstream of that in the “QM6a” model, and there is anon-sense mutation at the C-terminus (Poggi-Parodi et al., 2014),resulting a longer N-terminal sequence and shorter C-terminal proteinsequence (FIG. 1).

In the present Example, both the short ace3 ORF (based on the QM6aannotation, but including the RUT-C30 non-sense mutation that truncatesthe C-terminus of the protein (Ace3-S)) and the long ace3 ORF (based onthe RUT-C30 annotation (Ace3-L)) were cloned. As set forth in FIG. 1,both the short ace3 (Ace3-S) and long ace3 (Ace3-L) ORFs comprise theC-terminal non-sense mutation, as found in RUT-C30 (FIG. 1). To drivethe expression of the ace3 ORFs, a heterologous hexose kinase (hxk1)promoter and a heterologous pyruvate kinase (pki1) promoter were tested.

Thus, four (4) Ace3-expression vectors pYL1, pYL2, pYL3 and pYL4 (FIG.2A-2D) were constructed using standard molecular biological procedures.These expression vectors contain a vector backbone with the bacterialColE1 ori and AmpR gene for replication and selection in E. coli. Inaddition to the T. reesei pyr2 selection marker, a T. reesei promotersequence (i.e., promoters of hxk1 orpki1), and the ace3 ORF (ace3-L orace3-SC) with its native terminator are also present. The T. reeseipromoters and the ace3 ORFs were PCR amplified from T. reesei genomicDNA using Q5 High-fidelity DNA polymerase (New England Biolabs) and theprimers set forth below in Table 1.

The specific primers used to PCR amplify fragments for each vector arelisted as follows. To construct vector pYL1, the hxk1 promoter wasamplified using primer pair TP13 (SEQ ID NO: 7) and TP14 (SEQ ID NO: 8),the Ace3-L ORF was amplified using primers TP15 (SEQ ID NO: 9) and TP16(SEQ ID NO: 10), and the vector backbone was amplified using primer pairTP17 (SEQ ID NO: 11) and TP18 (SEQ ID NO: 12). The complete sequence ofplasmid pYL1 is provided as SEQ ID NO: 21.

To construct vector pYL2, the hxk1 promoter was PCR amplified usingprimer pair of TP13 (SEQ ID NO: 7) and TP19 (SEQ ID NO: 13), the Ace3-SCORF was amplified using primers TP20 (SEQ ID NO: 14) and TP16 (SEQ IDNO: 10), and the vector backbone was amplified using primer pair TP17(SEQ ID NO: 11) and TP18 (SEQ ID NO: 12). The complete sequence ofplasmid pYL2 is provided as SEQ ID NO: 22.

To construct vector pYL3, the pki1 promoter was PCR amplified usingprimer pair of TP21 (SEQ ID NO: 15) and TP22 (SEQ ID NO: 16), the Ace3-LORF was amplified using primers TP23 (SEQ ID NO: 17) and TP16 (SEQ IDNO: 10), and the vector backbone was amplified using primer pair TP17(SEQ ID NO: 11) and TP24 (SEQ ID NO: 18). The complete sequence ofplasmid pYL3 is provided as SEQ ID NO: 23.

To construct vector pYL4, the pki1 promoter was PCR amplified usingprimer pair of TP21 (SEQ ID NO: 15) and TP25 (SEQ ID NO: 19), theAce3-SC ORF was amplified using primers TP26 (SEQ ID NO: 20) and TP16(SEQ ID NO: 10), and the vector backbone was amplified using primer pairTP17 (SEQ ID NO: 11) and TP24 (SEQ ID NO: 18). The complete sequence ofplasmid pYL4 is provided as SEQ ID NO: 24.

For each vector, the three PCR fragments described above were assembledand transformed into NEB DH5a competent cells using Gibson assemblycloning kit (New England Biolabs; Catalogue No.: E5510S) according tomanufacturer's protocols. The resulting vectors were sequenced usingSanger sequencing, and their maps are shown in FIG. 2A-2D.

TABLE 1 Construct Assembly Primers Primer Sequence SEQ NO: TP13TCAGGGTTATTGTCTCATGGCCATTTAGGCCTGGCAGGCACTGGCTCGGACGACATGT  7 TP14AGAGCCCTGGGCCGGAGCTGCTGAGCCCATTGTTGAATTCTGGCGGGGTAGCTGTTGA  8 TP15TCAACAGCTACCCCGCCAGAATTCAACAATGGGCTCAGCAGCTCCGGCCCAGGGCTCT  9 TP16TCGTAAATAAACAAGCGTAACTAGCTAGCGTAGGTTATGCGAGCAACATTGCACGAAAC 10 TP17GTTTCGTGCAATGTTGCTCGCATAACCTACGCTAGCTAGTTACGCTTGTTTATTTACGA 11 TP18ACATGTCGTCCGAGCCAGTGCCTGCCAGGCCTAAATGGCCATGAGACAATAACCCTGA 12 TP19AGGTGTAAGACGGGGGAGTAGCGCAGCATTGTTGAATTCTGGCGGGGTAGCTGTTGA 13 TP20TCAACAGCTACCCCGCCAGAATTCAACAATGCTGCGCTACTCCCCCGTCTTACACCT 14 TP21TCAGGGTTATTGTCTCATGGCCATTTAGGCCTAGACTAGCGGCCGGTCCCCTTATCCCA 15 TP22AGAGCCCTGGGCCGGAGCTGCTGAGCCCATGGTGAAGGGGGCGGCCGCGGAGCCT 16 TP23AGGCTCCGCGGCCGCCCCCTTCACCATGGGCTCAGCAGCTCCGGCCCAGGGCTCT 17 TP24TGGGATAAGGGGACCGGCCGCTAGTCTAGGCCTAAATGGCCATGAGACAATAACCCTGA 18 TP25TGTAAGACGGGGGAGTAGCGCAGCATGGTGAAGGGGGCGGCCGCGGAGCCT 19 TP26AGGCTCCGCGGCCGCCCCCTTCACCATGCTGCGCTACTCCCCCGTCTTACA 201D. Transformation of T. reesei

The expression vectors of pYL1, pYL2, pYL3 and pYL4 were linearizedusing Pad enzyme (New England Biolabs), and transformed into T. reeseiparental host cells by polyethylene glycol (PEG)-mediated protoplasttransformation (Ouedraogo et al., 2015; Penttila et al., 1987). Thetransformants were grown on Vogel's minimal medium agar plates to selectfor uridine prototrophy acquired by the pyr2 marker. Stabletransformants were obtained by transfer on Vogel's agar plate for twosuccessive rounds, after which single colonies were obtained by platingdilution of spore suspension. The variant (i.e., modified) host cellsharboring pYL1, pYL2, pYL3 and pYL4 were named variant A4-7, variantB2-1, variant C2-28, and variant D3-1, respectively.

Example 2 Protein Production in Slow Release Microtiter Plate (srMTP)

The present example describes the screening method used to identifytransformants (see, Example 1) that secrete enzymes under non-inducingconditions. For example, stable transformants obtained from Example 1were tested in slow release microtiter plates (srMTP). The srMTP usedwere 24-well PDMS elastomer plates containing either 20% glucose (wt/wt)or 20% lactose (wt/wt), which were prepared as described in PCTInternational Publication No. WO2014/047520.

The parental and variant T. reesei host cells described in Example 1were tested under both “non-inducing” and “inducing” conditions. In the“non-inducing condition”, cells were grown in 1.25 ml liquid broth ofdefined medium, supplemented with 2.5% glucose (wt/vol) in a srMTPcontaining 20% glucose (wt/wt). In the “inducing condition”, cells weregrown in 1.25 ml liquid broth of defined medium supplemented with 2.5%glucose/sophorose (wt/vol) in a srMTP containing 20% lactose (wt/wt),where sophorose and lactose serve as potent inducers for cellulaseenzyme expression.

Preparation of glucose/sophorose was performed as described in U.S. Pat.No. 7,713,725. The defined medium was prepared as generally described inPCT International Publication No. WO2013/096056, comprising 9 g/Lcasamino acids, 5 g/L (NH₄)₂SO₄, 4.5 g/L KH₂PO₄, 1 g/L MgSO₄.7H₂O, 1 g/LCaCl₂.2H₂O, 33 g/L PIPPS buffer (at pH 5.5), 0.25 ml/L T. reesei traceelements. The T. reesei trace elements contains 191.41 g/l citricacid.H₂O, 200 g/L FeSO₄.7H₂O, 16 g/L ZnSO₄.7H₂O, 0.56 g/L CuSO₄.5H₂O,1.2 g/L MnSO₄.H₂O and 0.8 g/L H₃BO₃. All srMTP's were incubated at 28°C. for approximately 120 hours with continuous shaking at 280 rpm.

Following incubation, the supernatant from all cultures were harvestedand analyzed using Polyacrylamide Gel Electrophoresis (PAGE). Equalvolumes of culture supernatants were subjected to a reducing environmentfor fifteen (15) minutes at 90° C., before addition of loading dye andresolution on a 4-12% NuPage™ (Invitrogen, Carlsbad, Calif.)polyacrylamide gel with MOPS-SDS buffer. The gel was stained withSimplyBlue™ (Invitrogen) and imaged (see, FIG. 3).

As shown in FIG. 3, all of the host cells tested secreted a largequantity of proteins in the presence of an inducer (i.e., sophorose inthe media and lactose in the srMTP). However, in the absence of aninducer (i.e., sophorose or lactose), where glucose was the only carbonsource, only the variant host cells expressing the Ace3-L ORF (i.e.,variant A4-7 and variant C2-28) produced secreted proteins, while theparental host cells or variant cells expressing Ace3-SC ORF (i.e.,variant B1-1 and variant D3-1) did not produce extracellular proteinsabove the detection limit of PAGE. This result clearly demonstrates thatAce3-L (i.e., in contrast to Ace3-S) enables inducer-free proteinproduction in T. reesei.

The relative concentration of secreted proteins was determined by ZorbaxC3 reversed phase (RP) analysis using purified enzymes as a reference.For example, the secreted protein profiles of the host cells describedabove were analyzed using this method, wherein it was observed that allof the host cells (i.e., parental and variant cells) produced similarcellulase protein profiles under the inducing condition, wherein thecellulases consisted of approximately 40% CBH1, 20% CBH2, 10% EG1 and 7%of EG2. Under the non-inducing conditions, cellulase enzymes were belowdetection in the parental cells and the variant Ace3-S expressing hostcells. In contrast, it was surprisingly found that the variant Ace3-Lexpressing host cells (i.e., variants A4-7 and C2-28) produced a similarratio of cellulase enzymes as under the inducing conditions.

Briefly, this method of analysis was performed as follows: supernatantsamples were diluted in 50 mM sodium acetate buffer, pH 5.0, andde-glycosylated by addition of 20 ppm EndoH, incubated at 37° C. for 3hrs. Ten (10) μL 90% acetonitrile was added to 100 μL EndoH-treatedsample and passed over a 0.22 μm filter prior to injection. An Agilent1290 with DAD detection (Agilent Technologies) HPLC equipped with anAgilent Zorbax300 SB C3 RRHD 1.8 um (2.1×100 mm) column was used. Thecolumn was operating at 60° C. at a flow rate of 1.0 mL/min with 0.1%Trifluoroacetic acid (TFA) in MiliQ water as running buffer A and 0.07%TFA in Acetonitrile as running buffer B. The DAD detector was operatingat 220 nm and 280 nm with a 4 nm window. The injection volume was 10 μL.

Additionally, it was noted that the variant Ace3-L expressing host cellsproduced approximately 20-30% total extracellular proteins in the srMTPwith glucose (i.e., as compared to the total extracellular proteinsproduced in the srMTP with lactose). This relatively low expression maybe due to the high glucose feed rate in srMTP with glucose. For example,it is well established that the highest cellulase and hemi-cellulaseproduction rate is often observed with low growth rates (Arvas et al.,2011). Nevertheless, the srMTP growth assay was a relativelyhigh-throughput assay to screen stable colonies for protein production.

Taken together, the variant T. reesei host cells expressing the Ace3-LORF were able to produce cellulase and hemi-cellulase in the absence ofan inducer, albeit at a lower protein production rate. Moreparticularly, this low production rate was linked to the srMTP growthmethod, rather than the production ability of the host cells, as isshown in Example 3 and Example 4 below.

Example 3 Protein Production in Shake Flasks

To further explore and validate the srMTP results presented above, theparental host cell and variant Ace3-L expressing host cells were grownin the presence and absence of an inducer substrate in 50-mL submergedculture in shake flasks. More particularly, the parental T. reesei hostcells, the variant A4-7 cells and the variant C2-28 cells were grownunder both inducing conditions (i.e., glucose/sophorose as carbonsource) and non-inducing conditions (i.e., glucose as carbon source) insubmerged (liquid) culture and their respective extracellular (secreted)protein production levels were compared. Briefly, mycelia of each hostcell (i.e., the T. reesei parental host cell, the variant A4-7 host celland the variant C2-28 host cell) were added separately to 50-mL of YEGbroth in a 250-mL Erlenmeyer flask with bottom baffles. The YEG brothcontains 5 g/L yeast extract and 22 g/L glucose. The cell cultures weregrown for 48 hours, followed by sub-culturing into fresh YEG for another24 hours. These seed cultures were then inoculated into either 50 mL ofdefined medium supplemented with 1.5% glucose (non-inducing condition),or 50 mL of defined medium with 1.5% glucose/sophorose (inducingcondition) in 250 mL shake flasks with bottom baffles.

All shake flasks were incubated at 28° C. with continuous shaking at 200rpm. After 3 days of incubation, supernatant from all cell cultures wereharvested and analyzed using PAGE as described in Example 2 above. Thetotal protein in the supernatants were measured by the Bradforddye-binding assay at 595 nm using the Bio-Rad reagent (ThermoScientific®; Catalogue No.: 23236) and five dilutions of bovine serumalbumin (BSA) as a standard. The glucose concentrations were measured byHigh Performance Liquid Chromatography (HPLC) analysis, and no glucosewas detected in cultures after 3 days of incubation.

As shown in FIG. 4, the parental (control) T. reesei cells produced 464μg/mL total secreted protein in defined medium with glucose/sophorose(inducing), and only 140 μg/L of total secreted protein in definedmedium with glucose (non-inducing). In contrast, variant A4-7 cells andC2-28 cells produced similar amounts of secreted protein under inducingand non-inducing conditions, both of which are higher than the secretedprotein produced in the parental (control) cells with sophorose(induction). Thus, these results demonstrate that the variant cellsharboring the Ace3-L ORF (i.e., the variant A4-7 and C2-28 cells) notonly produce extracellular proteins in the absence of an inducer, butthese variant cells also produce more total protein than the parental(control) T. reesei cells under such inducing conditions.

Example 4 Protein Production in Small Scale Fed-Batch Fermentation

The instant example shows that the variant Ace3-L expressing cells(i.e., variant A4-7 and C2-28 cells) produced similar amounts ofcellulase and hemi-cellulase enzymes in the presence and absence of aninducer substrate in a small scale fermentation. More particularly, T.reesei fermentation was carried out generally as described in U.S. Pat.No. 7,713,725, using seed cultures in citrate minimal medium in a 2 Lbioreactor. More specifically, during fermentation, the supernatant fromall cultures was harvested at different time points, and equal volumesof the culture supernatants were subjected to PAGE analysis. As is shownin FIG. 5, the parental (control) T. reesei cells only produced secretedproteins in the presence of the sophorose inducer. In contrast, thevariant A4-7 and C2-28 cells (FIG. 5) produced similar amounts ofprotein, both under inducing and non-inducing conditions.

Example 5 Heterologous Promoters for Ace3 Expression

The present example demonstrates enhanced protein production under“non-inducing” conditions using thirteen (13) different promotersdriving the expression of ace3-L. More particularly, T. reesei cellsexpressing the ace3-L gene were generated by transforming parental T.reesei cells with a telomere vector containing the pyr2 gene, aheterologous promoter and the ace3-L gene, using protoplasttransformation.

Thus, thirteen T. reesei promoters were selected to drive the expressionof ace3-L ORF, wherein the thirteen promoters tested include, but arenot limited to: (i) a formamidase gene (rev3; Protein ID 103041)promoter (SEQ ID NO: 15), (ii) a β-xylosidase gene (bxl; Protein ID121127) promoter (SEQ ID NO: 16), (iii) a transketolase gene (tkl1;Protein ID 2211) promoter (SEQ ID NO: 17), (iv) a gene of unknownfunction (Protein ID 104295) promoter (SEQ ID NO: 18), (v) anoxidoreductase gene (dld1; Protein ID 5345) promoter (SEQ ID NO: 19),(vi) a xylanase IV gene (xyn4; Protein ID 111849) promoter (SEQ ID NO:20), (vii) an α-glucuronidase gene (Protein ID 72526) promoter (SEQ IDNO: 21), (viii) an acetyl xylan esterase gene 1 (axe1; Protein ID 73632)promoter (SEQ ID NO: 22), (ix) a hexose kinase gene (hxk1; Protein ID73665) promoter (SEQ ID NO: 23), (x) a mitochondrial carrier proteingene (dic1; Protein ID 47930) promoter (SEQ ID NO: 24), (xi) anoligopeptide transporter gene (opt; Protein ID 44278) promoter (SEQ IDNO: 25), (xii) a glycerol kinase gene (gut1; Protein ID 58356) promoter(SEQ ID NO: 26) and (xiii) a pyruvate kinase gene (pki1; Protein ID78439) promoter (SEQ ID NO: 27). Protein ID (PID) numbers are fromgenome.jgi.doe.gov/Trire2/Trire2.home.html. Thus, the thirteen promotersdescribed above were selected to drive expression because the genesthereof are generally expressed at a low level during growth whenglucose concentration is high, and are expressed at a higher level whenglucose concentration is low, or under sophorose-inducing conditions.

Table 2 below summarizes the thirteen promoters and expression vectorsthereof, which were constructed using standard molecular biologicalprocedures. More particularly, the expression vectors tested in theinstant example (Table 2) comprise a vector backbone with the bacterialColE1 ori and AmpR gene for replication and selection in E. coli, andthe 2μ ori and Ura3 gene for replication and selection in Saccharomycescerevisiae. In addition, T. reesei telomere sequences (“TrTEL”), T.reesei pyr2 selection marker, a T. reesei promoter sequence, and theace3-L ORF, with its native terminator sequence are present. Arepresentative vector map is shown in FIG. 7, depicting vector pYL8containing the dic1 promoter. Thus, the other vectors (e.g., pYL9,pYL12, etc.) have the same sequences presented in FIG. 7, except for thedifferent promoter sequences.

TABLE 2 ACE3-L EXPRESSION CONSTRUCTS UTILIZING DIFFERENT FUNGALPROMOTERS TO DRIVE ACE3-L EXPRESSION Vector # Promoter ace3 ORF pYL7 opt(SEQ ID NO: 25) ace3-L pYL8 dic1 (SEQ ID NO: 24) ace3-L pYL9 gut1 (SEQID NO: 26) ace3-L pYL12 hxk1 (SEQ ID NO: 23) ace3-L pYL13 pki1 (SEQ IDNO: 27) ace3-L pYL22 rev3 (SEQ ID NO: 15) ace3-L pYL23 PID 104295 (SEQID NO: 18) ace3-L pYL24 tkl1 (SEQ ID NO: 17) ace3-L pYL25 bxl (SEQ IDNO: 16) ace3-L pYL27 dld1 (SEQ ID NO: 19) ace3-L pYL28 xyn4 (SEQ ID NO:20) ace3-L pYL29 PID 72526 (SEQ ID NO: 21) ace3-L pYL30 axe1 (SEQ ID NO:22) ace3-L

The expression vectors were inserted (transformed) into a T. reeseiparental host strain (comprising a non-functional pyr2 gene) bypolyethylene glycol (PEG)-mediated protoplast transformation (Ouedraogoet al., 2015; Penttila et al., 1987). The transformants were grown onVogel's minimal medium agar plates to select for uridine prototrophyacquired by the pyr2 marker. Stable transformants were obtained bytransferring on Vogel's agar plate for two successive rounds, followedby two successive rounds of growth on non-selective PDA plates, and oneround on Vogel's agar plate, after which single colonies were obtainedby plating dilutions of a spore suspension.

The parental and transformed (daughter) T. reesei host cells describedabove were tested under both “non-inducing” and “inducing” conditions.For example, in the “non-inducing condition”, cells were grown in 1.25ml liquid broth of defined medium, supplemented with 2.5% glucose(wt/vol) in a regular 24 well microtiter plate (MTP). In the “inducingcondition”, cells were grown in 1.25 ml liquid broth of defined mediumsupplemented with 2.5% glucose/sophorose (wt/vol) in an MTP, whereinsophorose serves as a potent inducer for cellulase enzyme expression.Following incubation, the supernatants from all cultures were harvestedand the total secreted protein was measured by the Bradford dye-bindingassay at 595 nm using the Bio-Rad reagent (Thermo Scientific®; CatalogueNo.: 23236), and five dilutions of bovine serum albumin (BSA) as astandard.

As shown in Table 3, the parental T. reesei cells only produced highlevels of secreted proteins in the presence of the sophorose inducer. Incontrast, the variant (daughter) T. reesei cells, comprising andexpressing Ace3-L driven from thirteen different promoters, producedsimilar amounts of secreted protein, under both inducing andnon-inducing conditions. As shown in Table 3, the protein levels foreach modified (daughter) strain are presented as a ratio which isrelative to the protein (concentration) produced by the parental strain(LT4) under glucose/sophorose (Glu/Sop) inducing conditions.

TABLE 3 TOTAL SECRETED PROTEIN OF T. REESEI PARENTAL STRAIN (LT4)RELATIVE TO MODIFIED T. REESEI (DAUGHTER) STRAINS UNDER INDUCING(“GLU/SOP”) AND NON-INDUCING (“GLU”) CONDITIONS Strain ID PromoterGlu/Sop¹ Glu² LT4 (parental) N/A 1.00 0.20 LT82 opt 1.16 1.07 LT83 dic11.35 1.12 LT85 gut1 0.95 1.08 LT86 hxk1 0.96 0.73 LT87 pki1 1.16 0.98LT149 rev3 0.96 0.76 LT150 PID 104295 0.94 0.41 LT151 tkl1 1.02 1.01LT152 bxl 1.00 1.04 LT154 dld1 1.00 0.58 LT155 xyn4 0.95 0.95 LT156 PID72526 0.95 0.89 LT157 axe1 1.01 0.85 Glu/Sop¹ is an abbreviation of“Glucose/Sophorose”; Inducing Condition. Glu² is an abbreviation of“Glucose”; Non-Inducing Condition.

Additionally, the T. reesei parental strain and transformants thereofdescribed above were further tested in shake flasks experiments asgenerally described in Example 3. For example, a representative resultof T. reesei daughter strain “LT83” is shown in FIG. 8, wherein daughterstrain LT83 comprises an ace3-L ORF driven from a dic1 promoter (SEQ IDNO: 28). As shown in FIG. 8, the parental (control) T. reesei cells onlyproduced secreted proteins in the presence of the sophorose (“Sop”)inducer, whereas daughter strain LT83 produced similar amounts ofsecreted protein, under both inducing (“Sop”) and non-inducing (“Glu”)conditions.

Likewise, small scale fermentations were performed as generallydescribed in Example 4. More particularly, as shown in FIG. 9, theparental (control) T. reesei strain only produced secreted proteins inthe presence of the sophorose inducer (“Sop”), whereas daughter strainLT83 produced similar amounts of protein, under both inducing (“Sop”)and non-inducing (“Glu”) conditions.

Example 6 Cloning of T. reesei ACES Over Expression Constructs

Although the genome sequence of T. reesei is publicly available at theJoint Genome Institute (https://genome.jgi.doe.gov/), the position ofthe 5′ end of the ace3 coding region is not obvious. For example,annotation of the DNA sequence at the Joint Genome Institute differedbetween mutant strain Rut-C30(genome.jgi.doe.gov/TrireRUTC30_1/TrireRUTC30_1. home.html) and thewild-type strain QM6a (genome.jgi.doe.gov/Trire2/Trire2.home.html), eventhough the DNA sequence is the same. In the QM6a case, the 5′ end of theace3 coding region was suggested to be upstream (5′) of exon 3 andwithin intron 2, as shown in FIG. 11, arrow 3. In the Rut-C30 case, the5′ end of the ace3 coding region is within exon 2 (FIG. 11, arrow 2).Further analysis of the genomic DNA sequence and additional cDNAsequence suggested the possible existence of exon 1 and intron 1, asshown in FIG. 11. In addition, the 3′ end of the ace3 coding region inRut-C30 comprises a mutation, creating a premature stop codon (FIG. 11,arrow 4), relative to the sequence of the wild-type isolate QM6a (FIG.11, arrow 5). Thus, in the present example, the effects ofover-expressing these different possible forms of the ace3 gene wereexperimentally studied as described herein (e.g., see, FIG. 11, FIG. 12and FIG. 13-18).

Thus, the different forms of ace3 depicted in FIG. 12 wereover-expressed in T. reesei, wherein the over-expression vectors for theT. reesei ace3 genes were designed to enable targeted integration oface3 at the glucoamylase locus (gla1) in T. reesei. More particularly,in all of the plasmid (vector) constructs, the T. reesei ace3 gene wasexpressed under control of the T. reesei dic1 promoter and with thenative ace3 terminator. The vectors further comprise a pyr4 marker withits native promoter and terminator for selection of T. reeseitransformants A repeat of the pyr4 promoter was included to enableexcision of the pyr4 gene after integration at the gla1 locus. Thevector backbone enabling replication and amplification in E. coli wasEcoRI-XhoI digested pRS426 (Colot et al., 2006). The 5′ and 3′ flanks ofthe T. reesei gla1 locus needed for targeted integration, the dic1promoter, the different forms of the ace3 coding region and terminatorwere produced by PCR using primers given in Table 4. Template for theflanking fragments was genomic DNA from wild type T. reesei QM6a (ATCCDeposit No. 13631).

TABLE 4 PRIMERS USED TO GENERATE DNA FRAGMENTS PRIMER SEQUENCEGla.5F (SID: 49) GTAACGCCAGGGTTTTCCCAGTCACGACGGTTTAAACTCCATACGCAGCAAACATGGGCTTGGGC Gla.5R (SID: 50)GTACGAGTACTAGGTGTGAAGATTCCGTCAAGCTTGGGCGGAATGAAGGAGG ATGTGTGAGAGGDICprom.F (SID: 51) CACACATCCTCCTTCATTCCGCCCAAGCTTGACGGAATCTTCACACCTAGTACTCGTAC Ace3RutC.R (SID: 52)TGACATTTTTTGTTGTTCCAACACAGCATGCTTAGTCCGACGCCTTCGAGTC CAGCCAce3term.F (SID: 53)CTGGACTCGAAGGCGTCGGACTAAGCATGCTGTGTTGGAACAACAAAAAATG TCAce3term.R (SID: 54)GCAGAGCAGCAGTAGTCGATGCTATTAATTAAGTAGGTTATGCGAGCAACAT TG Gla.3F (SID: 55)CTCAGCCTCTCTCAGCCTCATCAGCCGCGGCCGCTGAATCGGCAAGGGGTAG TAC TAGGla.3R (SID: 56) GCGGATAACAATTTCACACAGGAAACAGCGTTTAAACCACATGCCAGAGTTCGATGCGCAAG Ace3_nointron.F (SID: 57)GTACCTCAGCGCTGTCGATAGCTGCACGCACTGCCGCGATGCCCACGTGCAG TGCACAce3_nointron.R (SID: 58)GTGCACTGCACGTGGGCATCGCGGCAGTGCGTGCAGCTATCGACAGCGCTGAG GTACTCAce3QM.F (SID: 59) GCGGCGCTTCCGCTGTCGTAACTATGCTGCGCTACTCCCCCGTCTTACDICprom_QM.R (SID: 60)GTAAGACGGGGGAGTAGCGCAGCATAGTTACGACAGCGGAAGCGCCGCCTTAT AAGTGAce3RutC.F (SID: 61)GGCGGCGCTTCCGCTGTCGTAACTATGGGCTCAGCAGCTCCGGCCCAGGGCTCDICprom_rutc.R (SID: 62)GCCCTGGGCCGGAGCTGCTGAGCCCATAGTTACGACAGCGGAAGCGCCGCCTT ATAAGAce3cDNA.F (SID: 63)GGCGGCGCTTCCGCTGTCGTAACTATGGCCACAGCGGCCGCGGCAGCAGCTGGDICprom_cDNA.R (SID: 64)CAGCTGCTGCCGCGGCCGCTGTGGCCATAGTTACGACAGCGGAAGCGCCGCCT TATAAG “SID” inthe above table is an abbreviation of “Sequence Identification Number”,e.g., “SEQ ID NO”

For the dic1 promoter, ace3 gene and terminator, template was an earlierplasmid pYL8 (See, Example 5) carrying these fragments. Selection marker(pyr4) was obtained from an earlier plasmid with NotI digestion. PCRprimers used to generate the desired DNA fragments are shown in Table 4.PCR products and digested fragments were separated using agarose gelelectrophoresis. Correct fragments were isolated from the gel with a gelextraction kit (Qiagen) according to manufacturer's protocol. Theplasmids were constructed with the fragments described above using yeasthomologous recombination method as described in PCT/EP2013/050126(published as WO2013/102674). Plasmids were rescued from yeast andtransformed to E. coli. A few clones were selected, plasmid DNA isolatedand sequenced. An overview of the plasmids is presented in Table 5.

TABLE 5 OVER-EXPRESSION PLASMIDS Gene SEQ Protein SEQ Plasmid CodeCloned gene Selection locus NOTE ID ID B7683 Sc ace3 SC form PYR4 GLAQM6a N-term; 7 8 RutC-30 C-term B7684 S ace3 S form PYR4 GLA QM6aN-term; 1 3 QM6a C-term B7709 L ace3 L form PYR4 GLA RutC-30 N-term; 4 6RutC-30 C-term B7752 LC ace3 LC form PYR4 GLA RutC-30 N-term; 9 10 QM6aC-term B7778 EL ace3 EL form PYR4 GLA RutC-30 N-term; 11 12 RutC-30C-term B7779 LN ace3 LN form PYR4 GLA RutC-30 N-term; 13 14 RutC-30C-term

Thus, the constructs presented in Table 5 differ by having differentforms of the ace3 gene. The SC form is a short form of the genecomprising exons 3 and 4, as well as intron 3 (see, FIG. 12 and FIG. 13,SEQ ID NO: 7). More particularly, SC form of SEQ ID NO: 7 comprises a1,713 bp Exon 3, a 148 bp Intron 3 and a 144 bp Exon 4. The (3′-end)C-terminus of the SC form (i.e., Exon 4) has the same mutation as the T.reesei RutC-30 strain.

The S form is a short form of the gene comprising exons 3 and 4, as wellas intron 3, but without the mutation in the (3′-end) C-terminus of Exon4 (see, FIG. 12 and FIG. 14, SEQ ID NO: 1). More particularly, the Sform comprises a 1,713 bp Exon 3, a 148 bp Intron 3 and a 177 bp Exon 4.In both of these forms (i.e., “SC” and “S”), the translation start codonis in the long intron 2 (see, FIG. 12), as annotated for T. reesei QM6astrain, and both “SC” and “S” forms are missing part of the codingregion for the putative DNA binding domain.

The L form is a long form of the gene comprising exons 2, 3 and 4, aswell as intron 2 (long intron) and intron 3 (e.g., see, FIG. 12 and FIG.15, SEQ ID NO: 4). More particularly, the L form comprises a 258 bp Exon2, a 423 bp Intron 2, a 1,635 bp Exon 3, a 148 bp Intron 3 and a 144 bpExon 4. The (3′-end) C-terminus of the “L” form has the same mutation asthe T. reesei RutC-30 strain.

The LC form is a long form of the gene comprising exons 2, 3 and 4, aswell as intron 2 (long intron) and intron 3 (e.g., see, FIG. 12 and FIG.16, SEQ ID NO: 9). More particularly, the LC form comprises a 258 bpExon 2, a 423 bp Intron 2, a 1,635 bp Exon 3, a 148 bp Intron 3 and a177 bp Exon 4. The LC form is without the mutation in the C-terminus. Inboth the “L” and “LC” forms, the translation start codon is within exon2 as annotated at JGI for the Rut-C30 strain.

The EL form is an extra-long version of the ace3 gene comprising exons1, 2, 3 and 4, as well as intron 1, intron 2 (long intron) and intron 3(e.g., see, FIG. 12 and FIG. 17, SEQ ID NO: 11). More particularly, theEL form comprises a 61 bp Exon 1, a 142 bp Intron 1, a 332 bp Exon 2, a423 bp Intron 2, a 1,635 bp Exon 3, a 148 bp Intron 3 and a 144 bp Exon4. The (3′-end) C-terminus of the EL form has the same mutation as theT. reesei RutC-30 strain.

The LN form is a long form of the gene containing exons 2, 3 and 4, aswell as intron 3, but lacking intron 2 (e.g., see, FIG. 12 and FIG. 18,SEQ ID NO: 13). The (3′-end) C-terminus of the LN form has the samemutation as the T. reesei RutC-30 strain. Thus, as described above, theL, LC, LN and EL forms of ace3 encode a full putative DNA bindingdomain.

Transformation into the T. reesei RL-P37 Strain

All of the plasmids presented in Table 5 were digested with MssI torelease the fragments for targeted integration and separated withagarose gel electrophoresis. For example, FIG. 19 provides a diagramshowing the arrangement of DNA sequences within a representativefragment used for transformation of T. reesei. Correct fragments wereisolated from the gel using a gel extraction kit (Qiagen) according tothe manufacturer's protocol. Approximately 10 μg purified fragment wasused to transform protoplasts of a pyr4⁻ mutant of T. reesei RL-P37strain. Preparation of protoplasts and transformation were carried outas described in PCT Publication No. WO2013/102674, using pyr4 selection.

Transformants were streaked onto selective medium plates. Growing cloneswere screened for correct integration by PCR using primers listed inTable 6. Clones giving expected signals were purified to single cellclones and rescreened for correct integration and clone purity by PCRusing primers listed in Table 6, as well as by Southern blotting (datanot shown).

TABLE 6 PCR PRIMERS Primer Sequence SEQ ID NO: Gla1_5creen.FGCTGGAAGCTGCTGAGCAGATC 65 DICprom.R GTGCCAGCATTCCCCAGACTCG 66T061_pyr4_orf_screen TTAGGCGACCTCTTTTTCCA 67 Gla1_3creen.RGCCGCTCAGGCATACGAGCGAC 68 DICprom.F2 CTCTGGTCGGCCTGCCGTTG 69 ace3.RTGAGTATAGCGGCTGACTTGTCG 70

Cultivation of the Different Ace3 Transformants

The strains in Table 7 were grown in 24-well microtiter plates in liquidmedium with either 2% lactose or 2% glucose as carbon source. The othercomponents of the medium were 0.45% KH2PO4, 0.5% (NH4)2SO4, 0.1% MgSO4,0.1% CaCl2, 0.9% Casamino acids, 0.048% Citric AcidxH2O, 0.05%FeSO4x7H2O, 0.0003% MnSO4xH2O, 0.004% ZnSO4x7H2O, 0.0002% H3BO3 and0.00014% CuSO4x5H2O. 100 mM PIPPS (Calbiochem) was included to maintainthe pH at 5.5.

TABLE 7 TRICHODERMA REESEI STRAINS Code Name of the strain SelectionM1904 RL-P37, parental strain M2015 ace3 SC clone 2-1 Pyr4 M2016 ace3 SCclone 28-3 Pyr4 M2017 ace3 S clone 9-1 Pyr4 M2018 ace3 S clone 20-1 Pyr4M2019 ace3 L clone 16-1 Pyr4 M2020 ace3 L clone 18-1 Pyr4 M2021 ace3 LCclone 52-1 Pyr4 M2022 ace3 LC clone 14-4 Pyr4 M2023 ace3 EL clone 3-3Pyr4 M2024 ace3 EL clone 4-5 Pyr4 M2025 ace3 LN clone 3-3 Pyr4 M2026ace3 LN clone 4-1 Pyr4

The cultures were carried out at 28° C. and 800 RPM in Infors HTmicroton shaker with 80% humidity. Sampling of the cultures wasperformed at days 3-7. The amount of total secreted proteins wasmeasured from the culture supernatants using Bio Rad Protein Assayaccording to manufacturer's protocol. In both media, the over-expressionof the ace3 L, EL and LN forms with the RutC-30 C-terminal mutation,improved the production of total proteins. In the medium with lactose asthe carbon source, over-expression of all the forms of ace3 geneimproved the production of total proteins to some extent, but the levelof improvement was highest in the strains over-expressing the L, EL andLN forms of the ace3 gene. It is clear that high levels of secretedprotein (Table 8) were observed when glucose (i.e., non-inducingcondition) was used as a carbon source with transformants in which theL, EL or LN forms of the ace3 gene were over-expressed.

TABLE 8 TOTAL PROTEINS PRODUCED BY THE DIFFERENT STRAINS IN 24-WELLPLATE CULTIVATION Strain 5d, mg/ml 7d, mg/ml LACTOSE M2015 0.77 1.21M2016 0.54 0.91 M2017 0.47 0.81 M2018 1.23 1.12 M2019 2.50 6.37 M20201.97 6.53 M2021 0.67 1.17 M2022 1.19 1.09 M2023 1.84 4.10 M2024 2.054.09 M2025 1.76 3.60 M2026 1.93 4.04 M1904 0.40 0.65 GLUCOSE M2015 0.590.89 M2016 0.12 0.56 M2017 0.00 0.19 M2018 0.02 0.26 M2019 1.57 2.74M2020 1.79 2.91 M2021 0.03 0.40 M2022 0.06 0.21 M2023 1.40 2.44 M20241.37 2.52 M2025 1.24 2.26 M2026 1.49 2.64 M1904 0.02 0.37

Example 7 Endogenous Ace3 Heterologous Promoter Knock-In

A promoter replacement construct (see, FIG. 6) is made by fusing a DNAfragment comprising a 5′ region upstream of the native promoter at theace3 locus, a loxP-flanked hygromycin B-resistance selectable markercassette, and a fragment comprising a promoter of interest operablyfused to the 5′ end of the ace3 open reading frame (e.g., seeInternational PCT Application Serial No. PCT/US2016/017113, whichfurther describes gene/promoter replacement cassettes for use infilamentous fungi).

Thus, a Trichoderma reesei cell is transformed with the promoterreplacement construct described above, wherein transformants areisolated and genomic DNA is extracted for diagnostic PCR to confirmhomologous recombination of the promoter replacement construct at thenative ace3 locus. Using this method, a transformant may be identifiedin which the native ace3 promoter is replaced by the hygromycin-Bresistance cassette and any promoter of interest. Subsequently, thehygromycin B-resistance cassette is removed by the action of crerecombinase (Nagy, 2000).

The efficiency of homologous integration at the ace3 locus may beenhanced by the action of cas9 directed to the native ace3 promoter by asuitably designed guide RNA as exemplified in International PCTPublication Nos: WO2016/100272, WO2016/100571 and WO2016/100568.

A conditional promoter replacement (CPR) strategy is described forAspergillus fumigatus in the publication by Hu et al. (2007), whichgenerally describes a strategy that uses the A. fumigatus NiiA nitrogenregulatable promoter (pNiiA) to delete and replace the endogenouspromoter of selected genes. Thus, in certain embodiments, an analogousmethod can be used to replace the endogenous promoter of the ace3 genein Trichoderma reesei with alternate promoters.

Example 8 Replacing an Endogenous Non-Lignocellulosic Gene of InterestPromoter with a Lignocellulosic Gene of Interest Promoter

A Trichoderma reesei glucoamylase expression construct was assembledfrom DNA polynucleotide fragments (e.g., see U.S. Pat. No. 7,413,879),wherein an ORF sequence encoding a T. reesei glucoamylase was operablylinked to a 5′ (upstream) T. reesei cbh1 promoter and operably linked toa 3′ (downstream) T. reesei cbh1 terminator. The DNA construct furthercomprised a T. reesei pyr2 gene as selectable marker.

Thus, a variant (daughter) T. reesei cell of the disclosure (i.e.,comprising a genetic modification which increases expression of a geneencoding an Ace3-L protein) was transformed with the glucoamylaseexpression construct. Transformants were selected and cultured in liquidmedium with glucose as carbon source (i.e., without an inducingsubstrate such as sophorose or lactose) in order to identify thosetransformants that were able to secrete the T. reesei glucoamylaseenzyme during culture.

Thus, a T. reesei glucoamylase expressing (parental) strain (control)and a modified T. reesei (daughter) glucoamylase expressing strain(i.e., comprising and expressing an Ace3-L ORF) were grown in shakeflask, and supernatant from all cell cultures were harvested andanalyzed using PAGE as generally described in Example 2 and Example 3above.

More particularly, as shown in FIG. 10, the parental T. reesei cellsproduced 1,029 μg/mL of glucoamylase in defined medium withglucose/sophorose (inducing condition), and only 38 μg/mL ofglucoamylase in defined medium with glucose (non-inducing condition). Incontrast, the modified (daughter) strain “LT88”, comprising ace3-Ldriven from the dic1 promoter, produced 3-fold higher glucoamylase under“inducing” (“Sop”) conditions (i.e., relative to the parental (control)strain under inducing conditions), and produced 2.5-fold higherglucoamylase under “non-inducing” (“Glu”) conditions (i.e., relative tothe parental (control) strain under inducing conditions) or 67-foldhigher glucoamylase under “non-inducing” (“Glu”) conditions relative tothe parental (control) strain under non-inducing conditions. Thus, theseresults demonstrate that the modified (daughter) cells comprising theAce3-L ORF not only produce extracellular proteins in the absence of aninducer, but these variant cells also produce more total protein thanthe parental (control) T. reesei cells under such inducing conditions.

Example 9 Replacing a Natively Associated Heterologous Gene of InterestPromoter with a Lignocellulosic Gene of Interest Promoter

A phytase expression construct is assembled from DNA polynucleotidefragments as follows (e.g., see U.S. Pat. No. 8,143,046). The ORFencoding Buttiauxella sp. phytase is operably linked at the 5′ end tothe T. reesei cbh1 promoter and at the 3′ end to the T. reesei cbh1terminator. The DNA construct further comprises a selectable marker, theAspergillus nidulans amdS gene. A variant T. reesei cell (i.e.,comprising a genetic modification which increases expression of a geneencoding an Ace3-L protein) is transformed with the phytase expressionconstruct. Transformants are selected and cultured in liquid medium withglucose as carbon source (i.e., without an inducing substrate such assophorose or lactose) in order to identify those transformants that areable to secrete Buttiauxella phytase enzyme during culture.

Example 10 Construction of Native Ace3 Promoter Replacement Vectors

In the present example, two ace3 promoter replacement vectors pCHL760and pCHL761 were constructed using standard molecular biologicalprocedures. Vector backbone pMCM3282 (FIG. 20) contained pMB1 ori andAmpR gene for replication and selection in E. coli. In addition, the hphhygromycin selection marker for Trichoderma reesei, expressed under N.crassa cpc1 promoter and A. nidulans trpC terminator, was included. Forpromoter replacement, the vectors contained a Streptococcus pyogenescas9 codon optimized for maize, expressed under T. reesei pki1 promoterand guide RNA expressed under U6 promoter (e.g., see, PCT PublicationNo: WO2016/100568 and WO2016/100272).

Thus, pMCM3282 was digested with EcoRV and 3 fragments having 5′ and 3′homology sequences of ˜1 kb from the T. reesei ace3 locus flankingeither the T. reesei hxk1 or dic1 promoter regions replacing the ace3native promoter, were cloned into pMCM3282/EcoRV by Gibson assembly,resulted in pCHL760 (FIG. 21) and pCHL761 (FIG. 22).

The 5′ and 3′ ace3 homology sequences, along with either the hxk1 ordic1 promoter were PCR amplified from T. reesei genomic DNA using Q5High-fidelity DNA polymerase (New England Biolabs) and the primers setforth below in Table 9.

TABLE 9 PCR PRIMERS CL1791 (SID: 71)TCTAGTATGTACGAGTACTAGGTGTGAAGATTCCGTCATTTCCTCGACAT GCGAATGCGCL1792 (SID: 72) TGCCATGCAAACCCCGCATTCGCATGTCGAGGAAATGACGGAATCTTCACACCTAGTAC CL1793 (SID: 73)TGCAGCTACAGAGCCCTGGGCCGGAGCTGCTGAGCCCATAGTTACGACA GCGGAAGCGCCL1794 (SID: 74) ATAGCACTTATAAGGCGGCGCTTCCGCTGTCGTAACTATGGGCTCAGCAGCTCCGGC CL1840 (SID: 75)TAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGGA TAGACTAGCATCTGAGCCATTGCAGC CL1786 (SID: 76)AGTGGCACCGAGTCGGTGGTGCTTTTTTTTCTATCGAGAGCATTGGTCAG TGGTGGCAAGCL1800 (SID: 77) ACCAATATACAAAACATGTCGTCCGAGCCAGTGCCTGCCATTTCCTCGACATGCGAATGC CL1801 (SID: 78)GTTGCCATGCAAACCCCGCATTCGCATGTCGAGGAAATGGCAGGCACTG GCTCGGACGACCL1802 (SID: 79) AGCTACAGAGCCCTGGGCCGGAGCTGCTGAGCCCATTGTTGAATTCTGGCGGGGTAGCTG CL1803 (SID: 80)CTTTTACACTTTTCAACAGCTACCCCGCCAGAATTCAACAATGGGCTCA GCAGCTCCGGCCL1831 (SID: 81) TAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGGTGCTTTTTTTTCTATCGAGATGTTCTGGATGGTGGAGAGG “SID” in the above table is anabbreviation of “Sequence Identification Number”, e.g., “SEQ ID NO”

More particularly, the specific primers used to PCR amplify fragmentsfor each vector are listed as follows. To construct pCHL760, 5′ upstreamhomology region was amplified using primer pair CL1840 and CL1791, 3′downstream homology region was amplified using primers CL1794 andCL1831, dic1 promoter was amplified using primer pair CL1792 and CL1793.

To construct pCHL761, 5′ upstream homology region was amplified usingprimer pair CL1840 and CL1800, 3′ downstream homology region wasamplified using primers CL1803 and CL1831, hxk1 promoter was amplifiedusing primer pair CL1801 and CL1802.

Vector pMCM3282 (FIG. 20) includes, from 5′ to 3′ direction, the T.reesei U6 promoter, an E. coli ccdB cassette, and the structural regionof a single-guide RNA (sgRNA) involved in Cas9 binding, including anintron from the U6 gene. The ccdB cassette was replaced with sequencesspecific for five different target sites within the Trichoderma ace3gene. Insertion of guide RNA sequences into pCHL760 (FIG. 21) andpCHL761 (FIG. 22) to construct final ace3 promoter replacement vectors.

Thus, the following oligonucleotides presented in Table 10 with Aar1restriction site were designed for production of different sgRNAsequences.

TABLE 10 Oligonucleotide sgRNA Sequences Oligo Oligonucleotide SEQ IDDescription Oligonucleotide Sequence ID NO: CL1821 top oligo for TS1AGTCTATCGCAGCCTTGCCTTAGCTAATGTTT 82 CL1822 bottom oligo for TS1TCTAAAACATTAGCTAAGGCAAGGCTGCGATA 83 CL1823 top oligo for TS4AGTCTATCGGCAGAGTCGCGTCTTCCGGGTTT 84 CL1824 bottom oligo for TS4TCTAAAACCCGGAAGACGCGACTCTGCCGATA 85 CL1825 top oligo for TS5AGTCTATCGAATGAGTGTAGGTACGAGTAGTTT 86 CL1826 bottom oligo for TS5TCTAAAACTACTCGTACCTACACTCATTCGATA 87 CL1827 top oligo for TS8AGTCTATCGGCCGCAATAGCTTCCTAATGTTT 88 CL1828 bottom oligo for TS8TCTAAAACATTAGGAAGCTATTGCGGCCGATA 89 CL1829 top oligo for TS10AGTCTATCGCAGCGCAATCAGTGCAGTGGTTT 90 CL1830 bottom oligo for TS10TCTAAAACCACTGCACTGATTGCGCTGCGATA 91

More particularly, CL1821 and CL1822, CL1823 and CL1824, CL1825 andCL1826, CL1827 and CL1828, CL1829 and CL1830 were annealed to createdouble stranded DNAs, which were cloned individually into pCHL760 andpCHL761 at AarI site using typeIIS seamless cloning method. The finalplasmids with correctly inserted guide RNA sequences lost the toxic ccdBgene.

Transformation of T. reesei

The cas9 mediated ace3 promoter replacement vectors of pCHL760 andpCHL761 were transformed into T. reesei parental cells by polyethyleneglycol (PEG)-mediated protoplast transformation. The transformants weregrown on Vogel's minimal medium agar with hygromycin to select forhygromycin resistant transformants. Some of these transformants wereunstable, having taken up the plasmid, but without stable integrationinto the genomic DNA. Transformants were transferred onto Vogel'snon-selective agar medium to allow loss of the plasmid andhygromycin-resistance marker.

To screen for dic1 promoter replaced transformants, genomic DNA wasextracted and PCR using primer pairs CL1858 and CL1848 (expected sizeproduct for desired integration was 2,412 bp), and CL1853 and CL1818(expected size product for desired integration was 2,431 bp), e.g. seeTable 11. PCR products were subsequently analyzed by DNA sequencing toconfirm the desired promoter integration.

To screen for hxk1 promoter replaced transformants, PCR using primerpairs CL1858 and CL1898 (expected size product for desired integrationwas 1,784 bp), and CL1853 and CL1850 (expected size product for desiredintegration was 2,178 bp) was performed and correct integrationsubsequently confirmed by DNA sequencing the PCR products, e.g. seeTable 11.

TABLE 11 PCR Primers Primer SEQ ID ID Primer Sequence NO: CL1858TGGAGAGACTCGGAGAGGATAGG 92 CL1853 AGCGTGGAGGCAGTTGGAGTGG 93 CL1848TGGACAAAGCCTGGGTCCTGCTCC 94 CL1818 ATCCTGACTCGTCCTGTGTCGG 95 CL1898AGTGCTTCGTTTAGTGGACTTG 96 CL1850 CTCGGTAGCTGCTTGAATATAG 97

Protein Production in Shake Flasks

To test the functionality of ace3 promoter replaced strains, cells weregrown in the presence and absence of an inducer substrate (sophorose) in50 ml submerged culture in shake flasks. The parental T. reesei hostcells (ID No. 1275.8.1) and the variant cells ID Nos. 2218, 2219, 2220,2221, 2222 and 2223, were grown under both inducing conditions(glucose/sophorose as carbon source) and non-inducing conditions(glucose as carbon source) in liquid culture, and their respectiveextracellular secreted protein production levels were compared. Briefly,mycelia of each host cell (i.e., the T. reesei parental host cell andthe variant cells thereof) were added separately to 50 mL of YEG brothin a 250 mL Erlenmeyer flask with bottom baffles. The YEG brothcontained 5 g/L yeast extract and 22 g/L glucose. The cell cultures weregrown for 48 hours, followed by sub-culturing into fresh YEG for another24 hours. These seed cultures were then inoculated into either 50 mL ofdefined medium supplemented with 2.5% glucose (non-inducing condition),or 50 mL of defined medium with 2.5% glucose/sophorose (inducingcondition) in 250 mL shake flasks with bottom baffles. All shake flaskswere incubated at 28° C. with continuous shaking at 200 rpm. After 4days of incubation, supernatant from all cell cultures were harvestedand analyzed using SDS-PAGE, as presented in FIG. 23.

As seen on above SDS-PAGE, parental cells (FIG. 23, ID 1275.8.1)produced much less secreted protein in defined medium with glucose(non-inducing) compared to glucose/sophorose (induction). In contrast,transformants 2218, 2219, 2220, 2222 and 2223 produced similar amountsof secreted protein under inducing and non-inducing conditions. Thus,these results demonstrate that the variant cells harboring the hxk1 ordic1 promoter replacing the native ace3 promoter at ace3 locus producedextracellular proteins in the absence of an inducer.

REFERENCES

-   European Patent Application No. EP 215,594-   European Patent Application No. EP 244,234-   European Publication No. EP 0215594-   PCT International Application Serial No. PCT/EP2013/050126-   PCT International Application Serial No. PCT/US2016/017113-   PCT International Publication No. WO03/027306-   PCT International Publication No. WO1992/06183-   PCT International Publication No. WO1992/06209-   PCT International Publication No. WO1992/06221-   PCT International Publication No. WO1992/10581-   PCT International Publication No. WO1998/15619-   PCT International Publication No. WO2002/12465-   PCT International Publication No. WO2003/52054-   PCT International Publication No. WO2003/52055-   PCT International Publication No. WO2003/52056-   PCT International Publication No. WO2003/52057-   PCT International Publication No. WO2003/52118-   PCT International Publication No. WO2004/16760-   PCT International Publication No. WO2004/43980-   PCT International Publication No. WO2004/48592-   PCT International Publication No. WO2005/001036-   PCT International Publication No. WO2005/01065-   PCT International Publication No. WO2005/028636-   PCT International Publication No. WO2005/093050-   PCT International Publication No. WO2005/28636-   PCT International Publication No. WO2005/93073-   PCT International Publication No. WO2006/074005-   PCT International Publication No. WO2006/74005-   PCT International Publication No. WO2009/149202-   PCT International Publication No. WO2010/141779-   PCT International Publication No. WO2011/038019-   PCT International Publication No. WO2011/063308-   PCT International Publication No. WO2011/153276-   PCT International Publication No. WO2012/125925-   PCT International Publication No. WO2012/125951-   PCT International Publication No. WO2012125937-   PCT International Publication No. WO2013/102674-   PCT International Publication No. WO2014/047520-   PCT International Publication No. WO2014/070837-   PCT International Publication No. WO2014/070841-   PCT International Publication No. WO2014/070844-   PCT International Publication No. WO2014/093275-   PCT International Publication No. WO2014/093281-   PCT International Publication No. WO2014/093282-   PCT International Publication No. WO2014/093287-   PCT International Publication No. WO2014/093294-   PCT International Publication No. WO2015/084596-   PCT International Publication No. WO2016/069541-   PCT International Publication No. WO2016/100272-   PCT International Publication No. WO2016/100568-   PCT International Publication No. WO2016/100571-   U.S. Pat. No. 6,022,725-   U.S. Pat. No. 6,268,328-   U.S. Pat. No. 7,413,879-   U.S. Pat. No. 7,713,725-   U.S. Pat. No. 8,143,046-   Alexopoulos, C. J., Introductory Mycology, New York: Wiley, 1962.-   Allen and Mortensen, Biotechnol. Bioeng., 2641-45, 1981.-   Arvas, M., Pakula, T., Smit, B., Rautio, J., Koivistoinen, H.,    Jouhten, P., Lindfors, E., Wiebe, M., Penttila, M., and Saloheimo,    M., “Correlation of gene expression and protein production rate-a    system wide study”, BMC Genomics 12, 616, 2011.-   Ausbel et al., “Current Protocols in Molecular Biology”, Green    Publishing Associates/Wiley Interscience, New York, 1987.-   Ausubel et al., Current Protocols in Molecular Biology, Green    Publishing Associates/Wiley Interscience, New York, 1994.-   Boel et al., EMBO J. 3:1581-1585, 1984.-   Campbell et al., Curr. Genet., 16: 53-56, 1989.-   Cao et al., Science, 9: 991-1001, 2000.-   Colot et al., PNAS 103(27):10352-10357, 2006.-   Devereux et al., Nucleic Acids Res. 12:387-395, 1984.-   el-Gogary et al., “Mechanism by which cellulose triggers    cellobiohydrolase I gene expression in Trichoderma reesei”, PNAS,    86(16)-6138-6141, 1989.-   Hakkinen, M., Valkonen, M. J., Westerholm-Parvinen, A., Aro, N.,    Arvas, M., Vitikainen, M., Penttila, M., Saloheimo, M., and    Pakula, T. M., “Screening of candidate regulators for cellulase and    hemicellulase production in Trichoderma reesei and identification of    a factor essential for cellulase production”, Biotechnol Biofuels 7,    14, 2014.-   Harkki et al., BioTechnol., 7: 596-603, 1989.-   Harkki et al., Enzyme Microb. Technol., 13: 227-233, 1991.-   Hu et al., “Essential gene identification and drug target    prioritization in Aspergillus fumigatus” PLoS Pathog., 3(3), 2007.-   Ilmen et al., “Regulation of cellulase gene expression in the    filamentous fungus Trichoderma reesei”, Applied and Environmental    Microbiology, 63(4)-1298-1306, 1997.-   Ju and Afolabi, Biotechnol. Prog., 91-97, 1999.-   Kriegler, Gene Transfer and Expression: A Laboratory Manual, 1990.-   Martinez et al., “Genome sequencing and analysis of the    bio-mass-degrading fungi Trichoderma reesei (syn. Hypocrea    jecorina)”, Nature Biotechnology, 26:533-560, 2008.-   Mullaney et al., MGG 199:37-45, 1985.-   Nagy, “Cre recombinase: the universal reagent for genome tailoring”,    Genesis 26(2), 99-109, 2000.-   Needleman and Wunsch, J. Mol. Biol., 48:443, 1970.-   Nunberg et al., Mol. Cell Biol. 4:2306, 1984.-   Ouedraogo, J. P., Arentshorst, M., Nikolaev, I., Barends, S., and    Ram, A. F., “I-SceI-mediated double-strand DNA breaks stimulate    efficient gene targeting in the industrial fungus Trichoderma    reesei” Applied microbiology and biotechnology 99, 10083-10095,    2015.-   Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988.-   Penttila, M., Nevalainen, H., Ratto, M., Salminen, E., and Knowles,    J., “A versatile transformation system for the cellulolytic    filamentous fungus Trichoderma reesei”, Gene 61, 155-164, 1987.-   Poggi-Parodi, D., Bidard, F., Pirayre, A., Portnoy, T., Blugeon, C.,    Seiboth, B., Kubicek, C. P., Le Crom, S., and Margeot, A., “Kinetic    transcriptome analysis reveals an essentially intact induction    system in a cellulase hyper-producer Trichoderma reesei strain”,    Biotechnol Biofuels 7, 173, 2014.-   Sambrook et al., Molecular Cloning, A Laboratory Manual, 2^(nd)    Edition, Cold Spring Harbor Laboratory Press, Cold Spring, New York,    1989.-   Sambrook et al., Molecular Cloning, A Laboratory Manual, 4^(th)    Edition, Cold Spring Harbor Laboratory Press, Cold Spring, New York,    2012.-   Seiboth, et. al., Mol. Genet. Genomics, 124-32, 2002.-   Sheir-Neiss and Montenecourt, “Characterization of the secreted    cellulases of Trichoderma reesei wild type and mutants during    controlled fermentations”, Applied Microbiology and Biotechnology,    20(1):46-53, 1984.-   Smith and Waterman, Adv. Appl. Math. 2:482, 1981.-   Vaheri et al., “Transglycosylation products of the cellulase system    of Trichoderma reesei”, Biotechnol. Lett., 1:41-46, 1979.-   Yelton et al., PNAS USA 81:1470-1474, 1984.

1-16. (canceled)
 17. A method for producing a lignocellulosic degradingenzyme in a Trichoderma sp. fungal cell in the absence of an inducingsubstrate comprising: introducing into the fungal cell a polynucleotideconstruct comprising an upstream (5′) promoter sequence operably linkedto a downstream (3′) nucleic acid encoding an Ace3 protein comprising atleast 95% sequence identity to SEQ ID NO: 6 and “Lys-Ala-Ser-Asp” as thelast four C-terminal amino acids, and fermenting the cell underconditions suitable for fungal cell growth and protein production,wherein such conditions do not include an inducing substrate.
 18. Themethod of claim 17, wherein the polynucleotide construct furthercomprises a downstream (3′) native ace3 terminator sequence operablylinked to the nucleic acid encoding the Ace3 protein.
 19. The method ofclaim 17, wherein the polynucleotide construct is integrated into thefungal cell genome.
 20. The method of claim 17, wherein thepolynucleotide construct is integrated into a telomere site of thefungal cell genome. 21-22. (canceled)
 23. The method of claim 17,wherein the encoded Ace3 protein comprises an amino acid sequencecomprising at least 95% sequence identity to SEQ ID NO: 6, SEQ ID NO: 12or SEQ ID NO:
 14. 24-57. (canceled)
 58. The method of claim 17, whereinthe lignocellulose degrading enzyme is selected from the groupconsisting of cellulase enzymes, hemi-cellulase enzymes, or acombination thereof.
 59. The method of claim 17, further comprising anintroduced expression cassette encoding a protein of interest.